Mutant Reverse Transcriptase and Methods of Use

ABSTRACT

The invention relates to the generation and characterization of stable MMLV reverse transcriptase mutants. The invention also discloses methods of using stable MMLV reverse transcriptase mutants.

This application is a Continuation of U.S. patent application Ser. No.11/502,819, filed Aug. 10, 2006, which claims the benefit of U.S.Provisional Application No. 60/707,019, filed on Aug. 10, 2005, thedisclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to mutant reverse transcriptases with increasedstability.

BACKGROUND

Three prototypical forms of retroviral reverse transcriptase have beenstudied thoroughly. Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase contains a single subunit of 78 kDa with RNA-dependent DNApolymerase and RNase H activity. This enzyme has been cloned andexpressed in a fully active form in E. coli (reviewed in Prasad, V. R.,Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, p. 135 (1993)). Human Immunodeficiency Virus (HIV)reverse transcriptase is a heterodimer of p66 and p51 subunits in whichthe smaller subunit is derived from the larger by proteolytic cleavage.The p66 subunit has both an RNA-dependent DNA polymerase and an RNase Hdomain, while the p51 subunit has only a DNA polymerase domain. ActiveHIV p66/p51 reverse transcriptase has been cloned and expressedsuccessfully in a number of expression hosts, including E. coli(reviewed in Le Grice, S. F. J., Reverse Transcriptase, Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory press, p. 163 (1993)).Within the HIV p66/p51 heterodimer, the 51-kD subunit is catalyticallyinactive, and the 66-kD subunit has both DNA polymerase and RNase Hactivity (Le Grice, S. F. J., et al., EMBO Journal 10:3905 (1991);Hostomsky, Z., et al., J. Virol. 66:3179 (1992)). Avian Sarcoma-LeukosisVirus (ASLV) reverse transcriptase, which includes but is not limited toRous Sarcoma Virus (RSV) reverse transcriptase, Avian MyeloblastosisVirus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV)Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis VirusMC29 Helper Virus MCAV reverse transcriptase, AvianReticuloendotheliosis Virus (REV-T) Helper Virus REV-A reversetranscriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reversetranscriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reversetranscriptase, Rous Associated Virus (RAV) reverse transcriptase, andMyeloblastosis Associated Virus (MAV) reverse transcriptase, is also aheterodimer of two subunits, alpha (approximately 62 kDa) and beta(approximately 94 kDa), in which alpha is derived from beta byproteolytic cleavage (reviewed in Prasad, V. R., Reverse Transcriptase,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p.135). ASLV reverse transcriptase can exist in two additionalcatalytically active structural forms, beta beta and alpha (Hizi, A. andJoklik, W. K., J. Biol. Chem. 252: 2281 (1977)). Sedimentation analysissuggests alpha beta and beta beta are dimers and that the alpha formexists in an equilibrium between monomeric and dimeric forms(Grandgenett, D. P., et al., Proc. Nat. Acad. Sci. USA 70:230 (1973);Hizi, A. and Joklik, W. K., J. Biol. Chem. 252:2281 (1977); and Soltis,D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA 85:3372 (1988)). TheASLV alpha beta. and beta beta reverse transcriptases are the only knownexamples of retroviral reverse transcriptase that include threedifferent activities in the same protein complex: DNA polymerase, RNaseH, and DNA endonuclease (integrase) activities (reviewed in Skalka, A.M., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1993), p. 193). The alpha form lacks the integrasedomain and activity.

The conversion of mRNA into cDNA by reverse transcriptase-mediatedreverse transcription is an essential step in many gene expressionstudies. However, the use of unmodified reverse transcriptase (RT) tocatalyze reverse transcription is inefficient for a number of reasons.First, reverse transcriptase sometimes degrades an RNA template beforethe first strand reaction is initiated or completed, primarily due tothe intrinsic RNase H activity present in reverse transcriptase. Inaddition, mis-priming of the mRNA template molecule can lead to theintroduction of errors in the cDNA first strand. RTs have in fact beenshown to incorporate one base error per 3000-6000 nucleotides for HIVRT, and 1/10,000 nucleotide for AMV RT during cDNA synthesis (Berger, S.L., et al., Biochemistry 22:2365-2372 (1983); Krug, M. S., and Berger,S. L., Meth. Enzymol. 152:316 (1987); Berger et al. Meth. Enzymol. 275:523 (1996)). Secondary structure of the mRNA molecule itself may makesome mRNAs refractory to first strand synthesis. Another factor whichinfluences the efficiency of reverse transcription is the ability of RNAto form secondary structures. Such secondary structures can form, forexample, when regions of RNA molecules have sufficient complementarityto hybridize and form double stranded RNA. Generally, the formation ofRNA secondary structures can be reduced by raising the temperature ofsolutions which contain the RNA molecules. Thus, in many instances, itis desirable to reverse transcribe RNA at temperatures above 37° C.However, art known reverse transcriptases generally lose activity whenincubated at temperatures much above 37° C. (e.g., 50° C.).

A variety of methods of attempting to engineer a thermostable reversetranscriptase are known in the art. These methods include usingthermostable DNA polymerases that contain reverse transcriptase activity(Shandilya et al., Extremophiles, 2004 8:243), mutagenizing thermostableDNA polymerases to increase their reverse transcriptase activity (U.S.2002/0012970), mutagenizing thermolabile reverse transcriptases (US2004/0209276), using Mn²⁺ instead of Mg²⁺ in the presence of Taq/Tth DNApolymerases (Myers et al., Biochemistry 1991 30:7661), and usingadditives such as trehalose with thermolabile reverse transcriptases(Carninci et al., 1999 Proc Natl Acad Sci USA 95:520).

Scientists in the field have also tried different enzyme compositionsand methods for increasing the fidelity of polymerization on DNA or RNAtemplates. For example, Shevelev et al., Nature Rev. Mol. Cell Biol.3:364 (2002) provides a review on 3′-5′ exonucleases. Perrino et al.,PNAS, 86:3085 (1989) reports the use of epsilon subunit of E. coli DNApolymerase III to increase the fidelity of calf thymus DNA polymerase a.Bakhanashvili, Eur. J. Biochem. 268:2047 (2001) describes theproofreading activity of p53 protein and Huang et al., Oncogene, 17:261(1998) describes the ability of p53 to enhance DNA replication fidelity.Bakhanashvili, Oncogene, 20:7635 (2001) also reports that p53 enhancesthe fidelity of DNA synthesis by HIV type I reverse transcriptase.Hawkins et al. describes the synthesis of full length cDNA from longmRNA transcripts (2002, Biotechniques, 34:768).

U.S. Patent Application 2003/0198944A1 and U.S. Pat. No. 6,518,019provide an enzyme mixture containing two or more reverse transcriptases(e.g., each reverse transcriptase having a different transcription pausesite) and optionally one or more DNA polymerases. U.S. PatentApplication 2002/0119465A1 discloses a composition that includes amutant thermostable DNA polymerase and a mutant reverse transcriptase(e.g., a mutant Taq DNA polymerase and a mutant MMLV-RT). U.S. Pat. No.6,485,917B1 and U.S. Patent application 2003/0077762 and EP patentapplication EP1132470 provide a method for synthesizing cDNA in thepresence of an enzyme having a reverse transcriptional activity and anα-type DNA polymerase having a 3′-5′ exonuclease activity.

Removal of the RNase H activity of reverse transcriptase can eliminatethe problem of RNA degradation of the RNA template and improve theefficiency of reverse transcription (Gerard, G. F., et al., FOCUS11(4):60 (1989); Gerard, G. F., et al., FOCUS 14(3):91 (1992)). Howeversuch reverse transcriptases (“RNase H-” forms) do not address theadditional problems of mis-priming and mRNA secondary structure.

There is a need in the art for a reverse transcriptase that exhibitsincreased stability.

SUMMARY OF THE INVENTION

The invention relates to the construction and characterization ofthermostable MMLV reverse transcriptase. The invention also relates tomethods of using the thermostable MMLV reverse transcriptase describedherein, as well as kits comprising this enzyme.

The invention relates to a mutant MMLV reverse transcriptase, wherein atleast one of the following amino acid positions comprises a mutation:E69, E302, W313, L435, N454 and M651.

The invention also relates to a mutant MMLV reverse transcriptase,comprising at least one of a glutamic acid to lysine mutation atposition E69, a glutamic acid to lysine mutation at position E302, aglutamic acid to arginine mutation at position E302, a tryptophan tophenylalanine mutation at position W313, a leucine to glycine mutationat position L435, a leucine to methionine mutation at position L435, anasparagine to lysine mutation at position N454, an asparagine toarginine mutation at position N454, and a methionine to leucine mutationat position M651.

The invention also relates to a mutant MMLV reverse transcriptase,selected from the group consisting of: E302R/E69K/W313F/L435G/N454K;E302R/W313F/L435G/N454K; E302R/W313F/L435G; E302R/E69K/N454K;E302R/W313F; and E69K/E302R/W313F/L435G/N454K/D524N.

In one embodiment, the mutant MMLV reverse transcriptase furthercomprises a C-terminal extension.

In another embodiment, the C-terminal extension is RDRNKNNDRRKAKENE.(SEQ ID NO: 1)

In another embodiment, the mutant MMLV reverse transcriptase lacks RNaseH activity.

In another embodiment, the mutant MMLV reverse transcriptase furthercomprises at least one of increased stability, increased accuracy,increased processivity, and increased specificity.

The invention also relates to an isolated polynucleotide comprising anucleotide sequence encoding a mutant MMLV reverse transcriptase,wherein at least one of the following amino acid positions comprises amutation: E69, E302, W313, L435, N454 and M651.

The invention also relates to an isolated polynucleotide comprising anucleotide sequence encoding a mutant MMLV reverse transcriptase,comprising at least one of a glutamic acid to lysine mutation atposition E69, a glutamic acid to lysine mutation at position E302, aglutamic acid to arginine mutation at position E302, a tryptophan tophenylalanine mutation at position W313, a leucine to glycine mutationat position L435, a leucine to methionine mutation at position L435, anasparagine to lysine mutation at position N454, an asparagine toarginine mutation at position N454, and a methionine to leucine mutationat position M651.

The invention also relates to an isolated polynucleotide comprising anucleotide sequence encoding a mutant MMLV reverse transcriptase,selected from the group consisting of: E302R/E69K/W313F/L435G/N454K;E302R/W313F/L435G/N454K; E302R/W313F/L435G; E302R/E69K/N454K;E302R/W313F; and E69K/E302R/W313F/L435G/N454K/D524N.

In one embodiment, the isolated polynucleotide, further encodes aC-terminal extension.

In one embodiment, the C-terminal extension is RDRNKNNDRRKAKENE. (SEQ IDNO: 1)

The invention also relates to a composition comprising a mutant MMLVreverse transcriptase, wherein at least one of the following amino acidpositions comprises a mutation: E69, E302, W313, L435, N454 and M651.

The invention also relates to a composition comprising a mutant MMLVreverse transcriptase, comprising at least one of a glutamic acid tolysine mutation at position E69, a glutamic acid to lysine mutation atposition E302, a glutamic acid to arginine mutation at position E302, atryptophan to phenylalanine mutation at position W313, a leucine toglycine mutation at position L435, a leucine to methionine mutation atposition L435, an asparagine to lysine mutation at position N454, anasparagine to arginine mutation at position N454, and a methionine toleucine mutation at position M651.

The invention also relates to a composition comprising a mutant MMLVreverse transcriptase, selected from the group consisting of:E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; andE69K/E302R/W313F/L435G/N454K/D524N.

In one embodiment, the mutant reverse transcriptase of the compositionfurther comprises a C-terminal extension.

In another embodiment, the C-terminal extension is RDRNKNNDRRKAKENE.(SEQ ID NO: 1)

In another embodiment, the mutant reverse transcriptase furthercomprises at least one of increased stability, increased accuracy,increased processivity, and increased specificity.

In another embodiment, the reverse transcriptase lacks RNase H activity.

In another embodiment, the composition further comprises an epsilonsubunit from an eubacteria.

In another embodiment, the epsilon subunit is from Eschericia coli.

In another embodiment, the epsilon subunit is epsilon 186 fromEschericia coli.

In another embodiment, the composition further comprises formamide,betaine or DMSO.

The invention also provides for a kit comprising a mutant MMLV reversetranscriptase, wherein at least one of the following amino acidpositions comprises a mutation: E69, E302, W313, L435, N454 and M651,and packaging materials thereof.

The invention also provides for a kit comprising a mutant MMLV reversetranscriptase, comprising at least one of a glutamic acid to lysinemutation at position E69, a glutamic acid to lysine mutation at positionE302, a glutamic acid to arginine mutation at position E302, atryptophan to phenylalanine mutation at position W313, a leucine toglycine mutation at position L435, a leucine to methionine mutation atposition L435, an asparagine to lysine mutation at position N454, anasparagine to arginine mutation at position N454, and a methionine toleucine mutation at position M651, and packaging materials thereof.

The invention also provides for a kit comprising a mutant MMLV reversetranscriptase, selected from the group consisting of:E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; andE69K/E302R/W313F/L435G/N454K/D524N, and packaging materials thereof.

In one embodiment, the mutant reverse transcriptase of the kit lacksRNase H activity.

In another embodiment, the mutant MMLV-reverse transcriptase of the kit,further comprises a C-terminal extension.

In another embodiment, the C-terminal extension is RDRNKNNDRRKAKENE.(SEQ ED NO: 1)

In another embodiment, the mutant reverse transcriptase of the kitfurther comprises at least one of increased stability, increasedaccuracy, increased processivity, and increased specificity.

In another embodiment, the kit further comprises an epsilon subunit froman eubacteria.

In another embodiment, the epsilon subunit is from Eschericia coli.

In another embodiment, the epsilon subunit is epsilon 186 fromEschericia coli. In another embodiment, the kit further comprisesformamide, betaine or DMSO.

The invention also provides for a method for cDNA synthesis comprisingproviding a mutant reverse transcriptase of the invention; andcontacting the mutant reverse transcriptase with a nucleic acid templateto permit cDNA synthesis.

The invention also provides for a method for cloning comprisingproviding a mutant reverse transcriptase of the invention; contactingthe mutant reverse transcriptase with a nucleic acid template togenerate a synthesized cDNA product and inserting the synthesized cDNAproduct into a cloning vector.

The invention also provides for a method for RT-PCR comprising:providing a mutant reverse transcriptase of the invention; andcontacting the mutant reverse transcriptase with a nucleic acid templateto replicate and amplify the nucleic acid template.

In one embodiment, the RT-PCR comprises end-point RT-PCR.

In another embodiment, the RT-PCR is performed in real-time.

The invention also provides for a method for cDNA library constructioncomprising providing a mutant reverse transcriptase of the invention;contacting the mutant reverse transcriptase with a nucleic acid templateto generate a synthesized cDNA product and inserting the synthesizedcDNA product into a vector.

The invention also provides for a method for preparing a microarraycomprising providing a mutant reverse transcriptase of the invention;contacting the mutant reverse transcriptase with a nucleic acid templateto generate a synthesized cDNA product and attaching the cDNA product toa substrate.

DEFINITIONS

As used herein, “reverse transcriptase activity” and “reversetranscription” refer to the ability of an enzyme to synthesize a DNAstrand (i.e. complementary DNA or cDNA) utilizing an RNA strand as atemplate. Reverse transcriptase activity may be measured by incubatingan enzyme in the presence of an RNA template and deoxynucleotides, inthe presence of an appropriate buffer, under appropriate conditions, forexample as described in Example 3.

As used herein, the term “reverse transcriptase (RT)” is used in itsbroadest sense to refer to any enzyme that exhibits reversetranscription activity as measured by methods disclosed herein or knownin the art. A “reverse transcriptase” of the present invention,therefore, includes reverse transcriptases from retroviruses, otherviruses, as well as a DNA polymerase exhibiting reverse transcriptaseactivity, such as Tth DNA polymerase, Taq DNA polymerase, Tne DNApolymerase, Tma DNA polymerase, etc. RT from retroviruses include, butare not limited to, Moloney Murine Leukemia Virus (M-MLV) RT, HumanImmunodeficiency Virus (HIV) RT, Avian Sarcoma-Leukosis Virus (ASLV) RT,Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, AvianErythroblastosis Virus (AEV) Helper Virus MCAV RT, AvianMyelocytomatosis Virus MC29 Helper Virus MCAV RT, AvianReticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian SarcomaVirus UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus Y73 Helper VirusYAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis AssociatedVirus (MAV) RT, and as described in U.S. Patent Application 2003/0198944(hereby incorporated by reference in its entirety). For review, see e.g.Levin, 1997, Cell, 88:5-8; Brosius et al., 1995, Virus Genes 11:163-79.Known reverse transcriptases from viruses require a primer to synthesizea DNA transcript from an RNA template. Reverse transcriptase has beenused primarily to transcribe RNA into cDNA, which can then be clonedinto a vector for further manipulation or used in various amplificationmethods such as polymerase chain reaction (PCR), nucleic acidsequence-based amplification (NASBA), transcription mediatedamplification (TMA), or self-sustained sequence replication (3 SR).

As used herein, the terms “reverse transcription activity” and “reversetranscriptase activity” are used interchangeably to refer to the abilityof an enzyme (e.g., a reverse transcriptase or a DNA polymerase) tosynthesize a DNA strand (i.e., cDNA) utilizing an RNA strand as atemplate. Methods for measuring RT activity are provided herein belowand also are well known in the art. For example, the Quan-T-RT assaysystem is commercially available from Amersham (Arlington Heights, Ill.)and is described in Bosworth, et al., Nature 1989, 341:167-168.

As used herein, the term “increased” reverse transcriptase activityrefers to the level of reverse transcriptase activity of a mutant enzyme(e.g., a mutant reverse transcriptase) as compared to its wild-typeform. A mutant enzyme is said to have an “increased” reversetranscriptase activity if the level of its reverse transcriptaseactivity (as measured by methods described herein or known in the art)is at least 10% or more than its wild-type form, for example, at least10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more or atleast 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold or more.

Reverse transcriptases of the invention include any reversetranscriptase having one or a combination of the properties describedherein. Such properties include, but are not limited to, enhancedstability, enhanced thermostability, reduced or eliminated RNase Hactivity, reduced terminal deoxynucleotidyl transferase activity,increased accuracy, increased processivity, increased specificity and/orincreased fidelity.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two polynucleotide strands or between two nucleotidesthrough base-pairing. It is known that an adenine nucleotide is capableof forming specific hydrogen bonds (“base pairing”) with a nucleotidewhich is thymine or uracil. Similarly, it is known that a cytosinenucleotide is capable of base pairing with a guanine nucleotide.

As used herein, “mutation” refers to a change introduced into a parentalor wild type DNA sequence that changes the amino acid sequence encodedby the DNA, including, but not limited to, substitutions, insertions,deletions, point mutations, mutation of multiple nucleotides or aminoacids, transposition, inversion, frame shift, nonsense mutations,truncations or other forms of aberration that differentiate thepolynucleotide or protein sequence from that of a wild-type sequence ofa gene or gene product. The consequences of a mutation include, but arenot limited to, the creation of a new character, property, function, ortrait not found in the protein encoded by the parental DNA, including,but not limited to, N terminal truncation, C terminal truncation orchemical modification. A “mutation” also includes an N- or C-terminalextension.

The present invention relates in particular to mutant or modifiedreverse transcriptases wherein one or more (e.g., one, two, three, four,five, ten, twelve, fifteen, twenty, etc.) amino acid changes have beenmade which renders the enzyme more stable in nucleic acid synthesis, ascompared to the unmutated or unmodified reverse transcriptases. As willbe appreciated by those skilled in the art, one or more of the aminoacids identified may be deleted and/or replaced with one or a number ofamino acid residues. In a preferred aspect, any one or more of the aminoacids may be substituted with any one or more amino acid residues suchas Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, and/or Val.

A reverse transcriptase of the present invention may have one or more ofthe following properties: (a) increased stability or increased half-lifeat elevated temperatures; (b) reduced, substantially reduced, or nodetectable RNase H activity, (c) reduced or substantially reducedterminal deoxynucleotidyl transferase activity, (d) increased accuracy,(e) increased specificity, (f) increased processivity and/or (d)increased fidelity. In some embodiments, a reverse transcriptase of theinvention may have a plurality of the properties listed above (e.g., areverse transcriptase may have enhanced thermostability, reduced RNase Hactivity, and enhanced accuracy). Reverse transcriptases of theinvention may have one or more of the following properties: (a)increased thermostability or increased half-life at elevatedtemperatures; (b) reduced, substantially reduced, or no detectable RNaseH activity, (c) reduced or substantially reduced terminaldeoxynucleotidyl transferase activity, and/or (d) increased fidelity.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. In contrast, the term “modified” or “mutant”refers to a gene or gene product which displays altered characteristicswhen compared to the wild-type gene or gene product. For example, amutant DNA polymerase in the present invention is a DNA polymerase whichexhibits a reduced uracil detection activity.

As used herein, “increased” refers to greater than 10% (e.g., 11%, 12%,13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or more), as compared to a wild-typeenzyme. “Increased” also refers to greater than at least 2-fold or more,(for example, 3, 4, 5, 10, 20, 50, 100, 1000, 10,000-fold or more), ascompared to a wild-type enzyme.

As used herein “stable” refers to exhibiting increased activity, asdefined herein, under denaturing conditions, including but not limitedto higher temperatures (for example greater than 37° C. (for example 38,39, 40, 50, 55, 60, 65, 70, 75, 80, 85° C. or more), or in the presenceof denaturing agents, including but not limited to DMSO or formamide orbetaine, as compared to the activity of a wild-type enzyme subjected toidentical denaturing conditions.

As used herein, “stable” includes “thermostable” as defined herein.

As used herein, “thermostable refers to an enzyme which is resistant toinactivation by heat. “Thermostable” also refers to an enzyme which isstable and active at temperatures as great as preferably between about38-100° C., for example 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100° C. and more preferably between about 40-80° C. (for example40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80° C.) to heat ascompared, for example, to a non-thermostable form of an enzyme with asimilar activity. Thermostable is further defined hereinbelow.

In one embodiment, the present invention provides a modified or mutatedreverse transcriptase having a reverse transcriptase activity that has ahalf-life of greater than that of the corresponding unmodified orun-mutated reverse transcriptase at an elevated temperature, i.e.,greater than 37° C. In some embodiments, the half-life of a reversetranscriptase of the present invention may be 5 minutes or greater andpreferably 10 minutes or greater at 50° C. In some embodiments, thereverse transcriptases of the invention may have a half-life (e.g., at50° C.) equal to or greater than about 25 minutes, preferably equal toor greater than about 50 minutes, more preferably equal to or greaterthan about 100 minutes, and most preferably, equal to or greater thanabout 200 minutes.

In some embodiments, the reverse transcriptases of the invention mayhave a half-life at 50° C. that is from about 10 minutes to about 200minutes, from about 10 minutes to about 150 minutes, from about 10minutes to about 100 minutes, from about 10 minutes to about 75 minutes,from about 10 minutes to about 50 minutes, from about 10 minutes toabout 40 minutes, from about 10 minutes to about 30 minutes, or fromabout 10 minutes to about 20 minutes.

Mutated or modified reverse transcriptases of the present invention mayhave a reverse transcriptase activity (e.g., RNA-dependent DNApolymerase activity) that has a longer half-life at 55° C. than thereverse transcriptase activity of a corresponding un-mutated orunmodified reverse transcriptase. At 55° C., the half-life of reversetranscriptase activity of a mutated or modified reverse transcriptase ofthe invention may be greater than about 2 minutes, greater than about 3minutes, greater than about 4 minutes, greater than about 5 minutes,greater than about 6 minutes, greater than about 7 minutes, greater thanabout 8 minutes, greater than about 10 minutes, greater than about 15minutes, greater than about 20 minutes, or greater than about 30minutes. At 55° C., the half-life of reverse transcriptase activity of areverse transcriptase of the invention may be from about 2 minutes toabout 60 minutes, from about 2 minutes to about 45 minutes, from about 2minutes to about 30 minutes, from about 2 minutes to about 20 minutes,from about 2 minutes to about 15 minutes, from about 2 minutes to about10 minutes, from about 2 minutes to about 8 minutes, from about 2minutes to about 7 minutes, from about 2 minutes to about 6 minutes,from about 2 minutes to about 5 minutes, from about 2 minutes to about 4minutes, or from about 2 minutes to about 3 minutes.

Reverse transcriptases of the present invention may produce more product(e.g., full length product) at elevated temperatures than other reversetranscriptases. In one aspect, comparisons of full length productsynthesis is made at different temperatures (e.g., one temperature beinglower, such as between 37° C. and 50° C., and one temperature beinghigher, such as between 50° C. and 78° C.) while keeping all otherreaction conditions similar or the same. The amount of full lengthproduct produced may be determined using techniques well known in theart, for example, by conducting a reverse transcription reaction at afirst temperature (e.g., 37° C., 38° C., 39° C., 40° C., etc.) anddetermining the amount of full length transcript produced, conducting asecond reverse transcription reaction at a temperature higher than thefirst temperature (e.g., 45° C., 50° C., 52.5° C., 55° C., etc.) anddetermining the amount of full length product produced, and comparingthe amounts produced at the two temperatures. A convenient form ofcomparison is to determine the percentage of the amount of full lengthproduct at the first temperature that is produced at the second (i.e.,elevated) temperature. The reaction conditions used for the tworeactions (e.g., salt concentration, buffer concentration, pH, divalentmetal ion concentration, nucleoside triphosphate concentration, templateconcentration, reverse transcriptase concentration, primerconcentration, length of time the reaction is conducted, etc.) arepreferably the same for both reactions. Suitable reaction conditionsinclude, but are not limited to, a template concentration of from about1 nM to about 1 μM, from about 100 nM to 1 μM, from about 300 nM toabout 750 nM, or from about 400 nM to about 600 nM, and a reversetranscriptase concentration of from about 1 nM to about 1 μM, from about10 nM to 500 nM, from about 50 nM to about 250 nM, or from about 75 nMto about 125 nM. The ratio of the template concentration to the reversetranscriptase concentration may be from about 100:1 to about 1:1, fromabout 50:1 to about 1:1, from about 25:1 to about 1:1, from about 10:1to about 1:1, from about 5:1 to about 1:1, or from about 2.5:1 to 1:1. Areaction may be conducted from about 5 minutes to about 5 hours, fromabout 10 minutes to about 2.5 hours, from about 30 minutes to about 2hours, from about 45 minutes to about 1.5 hours, or from about 45minutes to about 1 hour. A suitable reaction time is about one hour.Other suitable reaction conditions may be determined by those skilled inthe art using routine techniques and examples of such conditions areprovided below.

When the amount of full length product produced by a reversetranscriptase of the invention at an elevated temperature is compared tothe amount of full length product produced by the same reversetranscriptase at a lower temperature, at an elevated temperature, thereverse transcriptases of the invention may produce not less than about25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 100% of the amount of fulllength product produced at the lower temperature. In some cases, thereverse transcriptases of the invention may produce an amount of fulllength product at a higher temperature that is greater than the amountof full length product produced by the reverse transcriptase at a lowertemperature (e.g., 1% to about 100% greater). In one aspect, reversetranscriptases of the invention produce approximately the same amount(e.g., no more than a 25% difference) of full length product at thelower temperature compared to the amount of full length product made atthe higher temperature.

A reverse transcriptase of the present invention may be one thatsynthesizes an amount of full length product, wherein the amount of fulllength product synthesized at 50° C. is no less than 10% (e.g., fromabout 10% to about 95%, from about 10% to about 80%, from about 10% toabout 70%, from about 10% to about 60%, from about 10% to about 50%,from about 10% to about 40%, from about 10% to about 30%, or from about10% to about 20%) of the amount of full length product it synthesizes at40° C. In some embodiments, a reverse transcriptase of the invention isone wherein the amount of full length product synthesized at 50° C. isno less than 50% (e.g., from about 50% to about 95%, from about 50% toabout 80%, from about 50% to about 70%, or from about 50% to about 60%)of the amount of full length product it synthesizes at 40° C. In someembodiments, a reverse transcriptase of the invention is one wherein theamount of full length product synthesized at 50° C. is no less than 75%(e.g., from about 75% to about 95%, from about 75%, to about 90%, fromabout 75% to about 85%, or from about 75% to about 80%) of the amount offull length product it synthesizes at 40° C. In other embodiments, areverse transcriptase of the invention is one wherein the amount of fulllength product synthesized at 50° C. is no less than 85% (e.g., fromabout 85% to about 95%, or from about 85% to about 90%) of the amount offull length product it synthesizes at 40° C.

A reverse transcriptase of the invention may be one that synthesizes anamount of full length product, wherein the amount of full length productsynthesized at 52.5° C. is no less than 10% (e.g., from about 10% toabout 30%, from about 10% to about 25%, from about 10% to about 20%,from about 10% to about 15%, from about 20% to about 60%, from about 20%to about 40%, from about 20% to about 30%, from about 30% to about 80%,from about 30% to about 60%, from about 30% to about 45%, from about 40%to about 90%, from about 40% to about 80%, from about 40% to about 60%,from about 40% to about 50% from about 50% to about 90%, or from about50% to about 70%), of the amount of full length product it synthesizesat 40° C. In some embodiments, the amount of full length productsynthesized at 52.5° C. is no less than 30% (e.g., from about 30% toabout 70%, from about 30% to about 60%, from about 30% to about 50%, orfrom about 30% to about 40%) of the amount of full length product itsynthesizes at 40° C. In some embodiments, the amount of full lengthproduct synthesized at 52.5° C. is no less than 50% (e.g., from about50% to about 70%, from about 50% to about 65%, from about 50% to about60%, or from about 50% to about 55%), of the amount of full lengthproduct it synthesizes at 40° C.

A reverse transcriptase of the invention may be one that synthesizes anamount of full length product, wherein the amount of full length productsynthesized at 55° C. is no less than 1% (e.g., from about 1% to about30%, from about 1% to about 25%, from about 1% to about 20%, from about1% to about 15%, from about 1% to about 10%, or from about 1% to about5%) of the amount of full length product it synthesizes at 40° C. Insome embodiments, the amount of full length product synthesized at 55°C. is no less than 5% (e.g., from about 5% to about 30%, from about 5%to about to about 25%, from about 5% to about 20%, from about 5% toabout 15%, or from about 5% to about 10%) of the amount of full lengthproduct it synthesizes at 40° C. In some embodiments, the amount of fulllength product synthesized at 55° C. is no less than 10% (e.g., fromabout 10% to about 30%, from about 10% to about to about 25%, from about10% to about 20%, from about 10% to about 15%, from about 20% to about60%, from about 20% to about 40%, from about 20% to about 30%, fromabout 30% to about 80%, from about 30% to about 60%, from about 30% toabout 45%, from about 40% to about 90%, from about 40% to about 80%,from about 40% to about 60%, from about 40% to about 50% from about 50%to about 90%, or from about 50% to about 70%) of the amount of fulllength product it synthesizes at 40° C.

In another aspect, the reverse transcriptases of the invention arecapable of producing more nucleic acid product (e.g., cDNA) and,preferably, more full length product, at one or a number of elevatedtemperatures (typically between 40° C. and 78° C.) compared to thecorresponding un-mutated or unmodified reverse transcriptase (e.g., thecontrol reverse transcriptase). Such comparisons are typically madeunder similar or the same reaction conditions and the amount of productsynthesized by the control reverse transcriptase is compared to theamount of product synthesized by the reverse transcriptase of theinvention. Preferably, the reverse transcriptases of the inventionproduce at least about 5%, at least 10%, at least 15%, at least 25%, atleast 50%, at least 75%, at least 100%, or at least 200% more product orfull length product compared to the corresponding control reversetranscriptase under the same reaction conditions and temperature. Thereverse transcriptases of the invention preferably produce from about10% to about 200%, from about 25% to about 200%, from about 50% to about200%, from about 75% to about 200%, or from about 100% to about 200%more product or full length product compared to a control reversetranscriptase under the same reaction conditions and incubationtemperature. The reverse transcriptases of the invention preferablyproduce at least 2 times, at least 3 times, at least 4 times, at least 5times, at least 6 times, at least 7 times, at least 8 times, at least 9times, at least 10 times, at least 25 times, at least 50 times, at least75 times, at least 100 times, at least 150 times, at least 200 times, atleast 300 times, at least 400 times, at least 500 times, at least 1000times, at least 5,000 times, or at least 10,000 times more product orfull length product compared to a control reverse transcriptase (e.g.,the corresponding un-mutated or unmodified reverse transcriptase) underthe same reaction conditions and temperature. The reverse transcriptasesof the invention preferably produce from 2 to 10,000, 5 to 10,000, 10 to5,000, 50 to 5,000, 50 to 500, 2 to 500, 5 to 500, 5 to 200, 5 to 100,or 5 to 75 times more product or full length product than a controlreverse transcriptase under the same reaction conditions andtemperature.

In one aspect, the reverse transcriptases of the invention produce, at50° C., at least 25% more, preferably at least 50% more and morepreferably at least 100% more nucleic acid product or full lengthproduct than a control reverse transcriptase (which is preferably thecorresponding wild-type reverse transcriptase). In another aspect, at52.5° C., the reverse transcriptases of the invention produce at least1.5 times, at least 2 times, at least 2.5 times, at least 3 times, atleast 4 times, at least 5 times, at least 6 times, at least 7 times, atleast 8 times, at least 9 times, at least 10 times the amount of nucleicacid product or full length product compared to a control reversetranscriptase. In another aspect, at 55° C., the reverse transcriptasesof the invention produce at least 2 times, at least 5 times, at least 10times, at least 15 times, at least 20 times, at least 25 times, at least50 times, at least 75 times, at least 100 times the amount of nucleicacid product or full length product compared to a control reversetranscriptase. Such comparisons are preferably made under the samereaction conditions and temperature.

Modified or mutated reverse transcriptases of the present invention mayhave an increased thermostability at elevated temperatures as comparedto corresponding unmodified or un-mutated reverse transcriptases. Theymay show increased thermostability in the presence or absence an RNAtemplate. In some instances, reverse transcriptases of the invention mayshow an increased thermostability in both the presence and absence of anRNA template. Those skilled in the art will appreciate that reversetranscriptase enzymes are typically more thermostable in the presence ofan RNA template. The increase in thermostability may be measured bycomparing suitable parameters of the modified or mutated reversetranscriptase of the invention to those of a corresponding unmodified orun-mutated reverse transcriptase. Suitable parameters to compareinclude, but are not limited to, the amount of product and/or fulllength product synthesized by the modified or mutated reversetranscriptase at an elevated temperature compared to the amount orproduct and/or full length product synthesized by the correspondingun-modified or un-mutated reverse transcriptase at the same temperature,and/or the half-life of reverse transcriptase activity at an elevatedtemperature of a modified or mutated reverse transcriptase at anelevated temperature compared to that of a corresponding unmodified orun-mutated reverse transcriptase.

A modified or mutated reverse transcriptase of the invention may have anincrease in thermostability at 50° C. of at least about 1.5 fold (e.g.,from about 1.5 fold to about 100 fold, from about 1.5 fold to about 50fold, from about 1.5 fold to about 25 fold, from about 1.5 fold to about10 fold) compared, for example, to the corresponding un-mutated orunmodified reverse transcriptase. A reverse transcriptase of theinvention may have an increase in thermostability at 50° C. of at leastabout 10 fold (e.g., from about 10 fold to about 100 fold, from about 10fold to about 50 fold, from about 10 fold to about 25 fold, or fromabout 10 fold to about 15 fold) compared, for example, to thecorresponding un-mutated or unmodified reverse transcriptase. A reversetranscriptase of the invention may have an increase in thermostabilityat 50° C. of at least about 25 fold (e.g., from about 25 fold to about100 fold, from about 25 fold to about 75 fold, from about 25 fold toabout 50 fold, or from about 25 fold to about 35 fold) compared to acorresponding un-mutated or unmodified reverse transcriptase.

The present invention also contemplates a modified or mutatedthermostable reverse transcriptase, wherein the reverse transcriptasehas an increase in thermostability of greater than about 1.5 fold at52.5° C. (e.g., from about 1.5 fold to about 100 fold, from about 1.5fold to about 50 fold, from about 1.5 fold to about 25 fold, or fromabout 1.5 fold to about 10 fold) compared, for example, to thecorresponding un-mutated or unmodified reverse transcriptase. A reversetranscriptase of the invention may have an increase in thermostabilityat 52.5° C. of at least about 10 fold (e.g., from about 10 fold to about100 fold, from about 10 fold to about 50 fold, from about 10 fold toabout 25 fold, or from about 10 fold to about 15 fold) compared, forexample, to the corresponding un-mutated or unmodified reversetranscriptase. A reverse transcriptase of the invention may have anincrease in thermostability at 52.5° C. of at least about 25 fold (e.g.,from about 25 fold to about 100 fold, from about 25 fold to about 75fold, from about 25 fold to about 50 fold, or from about 25 fold toabout 35 fold) compared, for example, to the corresponding un-mutated orunmodified reverse transcriptase.

In other embodiments, the present invention provides a reversetranscriptase, wherein the reverse transcriptase has an increase inthermostability of greater than about 1.5 fold at 55° C. (e.g., fromabout 1.5 fold to about 100 fold, from about 1.5 fold to about 50 fold,from about 1.5 fold to about 25 fold, or from about 1.5 fold to about 10fold) compared to a corresponding un-mutated or unmodified reversetranscriptase. In some embodiments, a reverse transcriptase of theinvention may have an increase in thermostability at 55° C. of at leastabout 10 fold (e.g., from about 10 fold to about 100 fold, from about 10fold to about 50 fold, from about 10 fold to about 25 fold, or fromabout 10 fold to about 15 fold) compared to a corresponding un-mutatedor unmodified reverse transcriptase. In some embodiments, a reversetranscriptase of the invention may have an increase in thermostabilityat 55° C. of at least about 25 fold (e.g., from about 25 fold to about100 fold, from about 25 fold to about 75 fold, from about 25 fold toabout 50 fold, or from about 25 fold to about 35 fold) compared to acorresponding un-mutated or unmodified reverse transcriptase.

Nucleic acid templates suitable for reverse transcription according tothis aspect of the invention include any nucleic acid molecule orpopulation of nucleic acid molecules (preferably RNA and most preferablymRNA), particularly those derived from a cell or tissue. In a specificaspect, a population of mRNA molecules (a number of different mRNAmolecules, typically obtained from a particular cell or tissue type) isused to make a cDNA library, in accordance with the invention. Examplesof cellular sources of nucleic acid templates include bacterial cells,fungal cells, plant cells and animal cells.

As used herein, a “C-terminal extension” refers to a peptide tail ofrandom sequence. A C-terminal extension is preferably from 1 to 500amino acids, more preferably from 1 to 100, amino acids, and mostpreferably from 2 to 50 amino acids.

As used herein, “random” means relating to an amino acid sequence,wherein each amino acid of the sequence has an equal probability ofoccurring.

As used herein, “RNase H activity” refers to endoribonucleasedegradation of the RNA of a DNA-RNA hybrid to produce 5′ phosphateterminated oligonucleotides that are 2-9 bases in length. RNase Hactivity does not include degradation of single-stranded nucleic acids,duplex DNA or double-stranded RNA.

As used herein, the phrase “substantially lacks RNase H activity” meanshaving less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wildtype enzyme. The phrase “lacking RNase H activity” means havingundetectable RNase H activity or having less than about 1%, 0.5%, or0.1% of the RNase H activity of a wild type enzyme.

An enzyme with “reduced” RNase H activity is meant that the enzyme hasless than 50%, e.g., less than 40%, 30%, or less than 25%, 20%, morepreferably less than 15%, less than 10%, or less than 7.5%, and mostpreferably less than 5% or less than 2%, of the RNase H activity of thecorresponding wild type enzyme containing RNase H activity. The RNase Hactivity of an enzyme may be determined by a variety of assays, such asthose described, for example, in U.S. Pat. Nos. 5,405,776; 6,063,608;5,244,797; and 5,668,005 in Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988) and Gerard, G. F., et al., FOCUS 14(5):91 (1992), thedisclosures of all of which are fully incorporated herein by reference.

As used herein, “processivity” refers to the ability of a nucleic acidmodifying enzyme, for example a reverse transcriptase, to remainattached to the template or substrate and perform multiple modificationreactions. “Modification reactions” include but are not limited tosynthesis. “Processivity” also refers to the ability of a nucleic acidmodifying enzyme, for example a reverse transcriptase, to perform asequence of steps without intervening dissociation of the enzyme fromthe growing DNA chains. “Processivity” can depend on the nature of thenucleic-acid modifying enzyme, the sequence of a nucleic acid template,and reaction conditions, for example, salt concentration, temperature orthe presence of specific proteins.

As used herein, “increased processivity” refers to an increase of 5-10%,preferably 10-50%, more preferably 50-100% or more, as compared to awild type reverse transcriptase. Processivity and increased processivitycan be measured as described in Malboeuf et al., 2001, Biotechniques 30:1074.

As used herein, “accuracy” refers to “fidelity”, defined hereinbelow.Accuracy or fidelity can be measured as described in U.S. PatentApplication Nos. 60/559,810 and Ser. No. 11,100,183, incorporated byreference it their entirety herein.

As used herein, “specificity” refers to a decrease in the amount ofmispriming by the reverse transcriptase at the cDNA synthesis level whenthe reaction is performed at higher temperature, as compared to theamount of mispriming by a wild-type reverse transcriptase performingunder identical conditions. Specificity can be measured as described inMizuno Y, et al., Nucleic Acids Research, 1999, 27: 1345-1349.

As used herein, “decrease” refers to at least 1-fold or more, forexample, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,1000-fold or more, less than a wild-type enzyme performing underidentical conditions. “Decrease” also refers to at least 5% or more (forexample 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 99, 100%)less than a wild-type enzyme performing under identical conditions.

The term “fidelity,” as used herein, refers to the accuracy ofnucleotide synthesis by reverse transcriptase or template-dependent DNApolymerase, e.g., RNA-dependent or DNA-dependent DNA polymerase. Thefidelity of a DNA polymerase, including a reverse transcriptase, ismeasured by the error rate (the frequency of incorporating an inaccuratenucleotide, i.e., a nucleotide that is not incorporated in atemplate-dependent manner). The accuracy or fidelity of DNApolymerization is maintained by both the polymerase activity and the3′-5′ exonuclease activity. The term “high fidelity” refers to an errorrate equal to or lower than 33×10⁻⁶ per base pair (see Roberts J. D. etal., Science, 1988, 242: 1171-1173, the entirety hereby incorporated byreference). The fidelity or error rate of a DNA polymerase may bemeasured using assays known to the art (see for example, Lundburg etal., 1991 Gene, 108:1-6).

A reverse transcriptase having an “increased (or enhanced or higher)fidelity” is defined as a mutant or modified reverse transcriptase(including a DNA polymerase exhibiting reverse transcriptase activity)having any increase in fidelity compared to its wild type or unmodifiedform, i.e., a reduction in the number of misincorporated nucleotidesduring synthesis of any given nucleic acid molecule of a given length.Preferably there is 1.5 to 1,000 fold (more preferably 2 to 100 fold,more preferably 3 to 10 fold) reduction in the number of misincorporatednucleotides during synthesis of any given nucleic acid molecule of agiven length. For example, a mutated reverse transcriptase maymisincorporate one nucleotide in the synthesis of a nucleic acidmolecule segment of 1000 bases compared to an unmutated reversetranscriptase misincorporating 10 nucleotides in the same size segment.Such a mutant reverse transcriptase would be said to have a 10-foldincrease in fidelity.

As used herein, the terms “nucleic acid”, “polynucleotide” and“oligonucleotide” refer to primers, probes, and oligomer fragments to bedetected, and shall be generic to polydeoxyribonucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and toany other type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, or modified purine or pyrimidine bases (includingabasic sites). There is no intended distinction in length between theterm “nucleic acid”, “polynucleotide” and “oligonucleotide”, and theseterms will be used interchangeably. These terms refer only to theprimary structure of the molecule. Thus, these terms include double- andsingle-stranded DNA, as well as double- and single-stranded RNA.

As used herein, “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA) and deoxyribonucleotides are “incorporated” into DNA byDNA polymerases. The term nucleotide includes, but is not limited to,deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP,dTTP, or derivatives thereof. Such derivatives include, for example,[aS]dATP, 7-deaza-dGTP, 7-deaza-dATP, amino-allyl dNTPs, fluorescentlabeled dNTPs including Cy3, Cy5 labeled dNTPs. The term nucleotide asused herein also refers to dideoxyribonucleoside triphosphates (ddNTPsand acyclic nucleotides) and their derivatives (e.g., as described inMartinez et al., 1999, Nucl. Acids Res. 27: 1271-1274, herebyincorporated by reference in its entirety).

As used herein, a “primer” refers to a sequence of deoxyribonucleotidesor ribonucleotides comprising at least 3 nucleotides. Generally, theprimer comprises from about 3 to about 100 nucleotides, preferably fromabout 5 to about 50 nucleotides and even more preferably from about 5 toabout 25 nucleotides. A primer having less than 50 nucleotides may alsobe referred to herein as an “oligonucleotide primer”. The primers of thepresent invention may be synthetically produced by, for example, thestepwise addition of nucleotides or may be fragments, parts, portions orextension products of other nucleotide acid molecules. The term “primer”is used in its most general sense to include any length of nucleotideswhich, when used for amplification purposes, can provide a free 3′hydroxyl group for the initiation of DNA synthesis by a DNA polymerase,either using an RNA or a DNA template. DNA synthesis results in theextension of the primer to produce a primer extension productcomplementary to the nucleic acid strand to which the primer hashybridized.

As used herein, the term “homology” refers to the optimal alignment ofsequences (either nucleotides or amino acids), which may be conducted bycomputerized implementations of algorithms. “Homology”, with regard topolynucleotides, for example, may be determined by analysis with BLASTNversion 2.0 using the default parameters. “Homology”, with respect topolypeptides (i.e., amino acids), may be determined using a program,such as BLASTP version 2.2.2 with the default parameters, which alignsthe polypeptides or fragments being compared and determines the extentof amino acid identity or similarity between them. It will beappreciated that amino acid “homology” includes conservativesubstitutions, i.e. those that substitute a given amino acid in apolypeptide by another amino acid of similar characteristics. Typicallyseen as conservative substitutions are the following replacements:replacements of an aliphatic amino acid such as Ala, Val, Leu and Ilewith another aliphatic amino acid; replacement of a Ser with a Thr orvice versa; replacement of an acidic residue such as Asp or Glu withanother acidic residue; replacement of a residue bearing an amide group,such as Asn or Gln, with another residue bearing an amide group;exchange of a basic residue such as Lys or Arg with another basicresidue; and replacement of an aromatic residue such as Phe or Tyr withanother aromatic residue. A polypeptide sequence (i.e., amino acidsequence) or a polynucleotide sequence comprising at least 50% homologyto another amino acid sequence or another nucleotide sequencerespectively has a homology of 50% or greater than 50%, e.g., 60%, 70%,80%, 90% or 100% (i.e., identical).

The term “E. coli DNA polymerase III holoenzyme” refers to an E. colipolymerase III holoenzyme composed of ten subunits assembled in twocatalytic cores, two sliding clamps and a clamp loader, e.g., asdescribed in Kelman, Z. & O'Donnell, M. (1995). Annu. Rev. Biochem. 64,171200 (the entirety is hereby incorporated by reference).

The term “epsilon (c) subunit,” according to the present invention,refers to a c subunit having 3′-5′ exonuclease activity. An epsilonsubunit may be from any eubacteria, such as from E. coli, or from otherorganisms. The epsilon (c) subunit of the E. coli DNA polymerase IIIholoenzyme is the 3′-5′ exonuclease of the holoenzyme and interacts withthe a (polymerase unit) and θ (unknown function) subunits (see, e.g.,Fijalkowska et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 2856-2861, theentirety is hereby incorporated by reference). The epsilon (c) subunitof E. coli DNA polymerase III holoenzyme (see FIG. 9) is encoded by dnaQgene (see FIG. 9). The epsilon subunit of the present invention alsoincludes a wild type polypeptide which is at least 50% homologous (e.g.,60%, 70%, 80%, 90%, or identical) to the sequences presented in FIG. 9and contains 3′-5′ exonuclease activity. The epsilon (c) subunit,according to the present invention, further includes a mutant epsilon(c) subunit which still contains 3′-5′ exonuclease activity. Such mutantepsilon may contain a deletion (e.g., truncation), substitution, pointmutation, mutation of multiple amino acids, or insertion to the wildtype epsilon subunit. For example, a truncated epsilon useful accordingto the invention may be, for example, as disclosed in Hamdan S. et al.,Biochemistry 2002, 41: 5266-5275, the entirety hereby incorporated byreference.

As used herein, “epsilon 186” refers to the N-terminal domain of theepsilon subunit (codons 2-186 of dnaQ), as disclosed in Hamdan et al.,supra.

The term “θε subunit complex” or “θε 186 subunit complex” refers to thecombination of the epsilon subunit or a mutant epsilon subunit of E.coli DNA polymerase III holoenzyme (for example epsilon 186) incombination with the θ subunit of E. coli DNA polymerase III holoenzyme.

As used herein, the term “eubacteria” refers to unicelled organismswhich are prokaryotes (e.g., as described in Garrity, et al., 2001,Taxonomic outline of the procaryotic genera. Bergey's Manual® ofSystematic Bacteriology, Second Edition. Release 1.0, April 2001, and inWerren, 1997, Annual Review of Entomology 42: 587-609). Eubacteriainclude the following genera: Escherichia, Pseudomonas, Proteus,Micrococcus, Acinetobacter, Klebsiella, Legionella, Neisseria,Bordetella, Vibrio, Staphylococcus, Lactobaccilus, Streptococcus,Bacillus, Corynebacteria, Mycobacteria, Clostridium, and others (seeKandler, O., Zbl. Bakt. Hyg., I. Abt. Orig. C3, 149-160 (1982)), as wellas major sub-groups of eubacteria such as Aquifex (extremelythermophilic chemolithotrophs), Thermotoga (extremely thermophilicchemoorganotrophs), Chloroflexus (thermophilic photosynthetic bacteria),Deinococcus (radiation resistant bacteria), Thermus (thermophilicchemoheterotrophs), Spirochaetes (helical bacteria with periplasmicflagella), Proteobacteria (Gram-negative and purple photosyntheticbacteria), Cyanobacteria (blue-green photosynthetic bacteria),Gram-positives (Gram-positive bacteria), Bacteroides/Flavobacterium(strict anaerobes/strict aerobes with gliding motility), Chlorobium(photoautotrophic sulphur-oxidisers), Planctomyces (budding bacteriawith no peptidoglycan), Chlamydia (intracellular parasites). Eubacteriainclude all thermostable bacteria.

As used herein, “synthesis” refers to any in vitro method for making anew strand of polynucleotide or elongating existing polynucleotide(i.e., DNA or RNA) in a template dependent manner. Synthesis, accordingto the invention, may include amplification, which increases the numberof copies of a polynucleotide template sequence with the use of apolymerase. Polynucleotide synthesis results in the incorporation ofnucleotides into a polynucleotide (i.e., a primer), thereby forming anew polynucleotide molecule complementary to the polynucleotidetemplate. The formed polynucleotide molecule and its template can beused as templates to synthesize additional polynucleotide molecules.

As used herein, an “amplified product” refers to the single- ordouble-strand polynucleotide population at the end of an amplificationreaction. The amplified product contains the original polynucleotidetemplate and polynucleotide synthesized by DNA polymerase using thepolynucleotide template during the amplification reaction. An amplifiedproduct preferably is produced by a reverse transcriptase and/or a DNApolymerase.

As used herein, “polynucleotide template” or “target polynucleotidetemplate” refers to a polynucleotide (RNA or DNA) which serves as atemplate for a DNA polymerase to synthesize DNA in a template-dependentmanner. The “amplified region,” as used herein, is a region of apolynucleotide that is to be either synthesized by reverse transcriptionor amplified by polymerase chain reaction (PCR). For example, anamplified region of a polynucleotide template may reside between twosequences to which two PCR primers are complementary to.

As used herein, the term “template dependent manner” refers to a processthat involves the template dependent extension of a primer molecule(e.g., DNA synthesis by DNA polymerase). “Template dependent manner”refers to polynucleotide synthesis of RNA or DNA wherein the sequence ofthe newly synthesized strand of polynucleotide is dictated by thewell-known rules of complementary base pairing (see, for example,Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A.Benjamin, Inc., Menlo Park, Calif. (1987)).

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis, e.g., as described in U.S. Pat. No.4,683,195 4,683,202, and 4,965,188 (each hereby incorporated in itsentirety by reference) and any other improved method known in the art.PCR is a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence typicallyconsists of introducing a large excess of two oligonucleotide primers tothe DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified”.

As used herein, the term “RT-PCR” refers to the replication andamplification of RNA sequences. In this method, reverse transcription iscoupled to PCR, e.g., as described in U.S. Pat. No. 5,322,770, hereinincorporated by reference in its entirety. In RT-PCR, the RNA templateis converted to cDNA due to the reverse transcriptase activity of anenzyme, and then amplified using the polymerizing activity of the sameor a different enzyme. Stable, thermostable or thermolabile reversetranscriptase and polymerase can be used.

Amino acid residues identified herein are preferred in the naturalL-configuration. In keeping with standard polypeptide nomenclature, J.Biol. Chem., 243:3557-3559, 1969, abbreviations for amino acid residuesare as shown in the following Table 1.

TABLE 1 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine G Gly glycine FPhe L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine IIle L-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P ProL-proline K Lys L-lysine H His L-histidine Q Gln L-glutamine E GluL-glutamic acid W Trp L-tryptophan R Arg L-arginine D Asp L-asparticacid N Asn L-asparagine C Cys L-cysteine

A “double tube RT-PCR” or “two step RT-PCR” refers to a reaction whereinthe RT step is performed in a first tube, and then the cDNA istransferred to a second tube for amplification. Therefore, the cDNAsynthesis and PCR occur in two separate tubes. A “single tube RT-PCR” or“one step RT-PCR” refers to a reaction wherein both cDNA synthesis andPCR are performed in the same tube.

As used herein, “end-point RT-PCR” refers to RT-PCR wherein a templateis added at the beginning of a PCR reaction and the reaction is carriedout in multiple cycles, usually 20 to 50 cycles. It is the end productof the amplification reaction which is detected and/or quantitated.

As used herein, “real time RT-PCR” or “quantitative” or “QRT-PCR” refersto an RT-PCR process wherein the progress of an RT-PCR amplification ismeasured or detected as it is occurring. In real-time RT-PCR techniques,signals (generally fluorescent) are monitored as they are generated andare tracked after they rise above background but before the reactionreaches a plateau.

As used herein, the term “real-time” refers to that which is performedcontemporaneously with the monitored, measured or observed events andwhich yields as a result of the monitoring, measurement or observationto one who performs it simultaneously, or effectively so, with theoccurrence of a monitored, measured or observed event. Thus, a “realtime” assay or measurement contains not only the measured andquantitated result, such as generated signal, but expresses this in realtime, that is, in hours, minutes, seconds, milliseconds, nanoseconds,picoseconds, etc.

As used herein, “microarray” refers to a plurality of nucleic acidmembers stably associated with a substrate. The term “array” is usedinterchangeably with the term “microarray,” however, the term“microarray” is used to define an array which has the additionalproperty of being viewable microscopically.

As used herein, “viewable microscopically” refers to an object which canbe placed on the stage of a dissecting or compound microscope andcomprises at least a portion which can be viewed using an ocular of themicroscope.

As used herein, “stably associated” refers to an association with aposition on a substrate that does not change under nucleic acidhybridization and washing conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of an MMLV-RT thermostability screen.

FIG. 2 presents the thermostability of His-tagged purified MMLV-RT pointmutants.

FIG. 3 presents the thermostability of C-terminally extended mutants.HSRRRLKRHIFN=SEQ ID NO:64; SKRTNPINIHTNK=SEQ ID NO:65; QEGKNRQGEGQT=SEQID NO:66; RDRNKNNDRRKAKENE=SEQ ID NO:1; RDRNKNNDRRKAKRDRNKNNDRRKAK=SEQID NO:67; RDRNKNNDRRKAKENEENEENEENEENE=SEQ ID NO:68.

FIG. 4 presents the results of an activity assay for an RT comprisingmultiple mutations. RFGK=SEQ ID NO:37; RKFGK=SEQ ID NO:36.

FIG. 5 presents cDNA ladder synthesis by His-tagged RTs. RKFGK=SEQ IDNO:36.

FIG. 6 presents the thermostability of RTs of the invention.RDRNKNNDRRKAKENE=SEQ ID NO:1.

FIGS. 7A, 7B, and 7C represent the half-life of mutant RTs according tothe invention.

FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 8I, 8J, 8K, 8L, 8M, 8N, 80, 8P,8Q, 8R, 8S, 8T, 8U, 8V, 8W, 8X, 8Y, 8Z, 8AA, 8BB, 8CC, 8DD, 8EE, 8FF,8GG, and 8HH present the nucleic acid and amino acid sequences of themutant RTs according to the invention. Optional codons are representedby XXX in FIGS. 8B, 8C, 8D, 8E, 8F, 8G, 8H, 8I, 8J, 8K, 8L, 8M, 8N, 80,8P, and 8Q. Possible nucleic acids sequences for XXX are presented abovethe XXX. Mutant amino acids are circled in FIGS. 8S, 8T, 8U, 8V, 8W, 8X,8Y, 8Z, 8AA, 8BB, 8CC, 8DD, 8EE, 8FF, 8GG, and 8HH.

FIG. 8A. WT-MMLV-RT nucleic acid sequence (SEQ ID NO: 2)

FIG. 8B. MMLV-RT E69K nucleic acid sequence (SEQ ID NO: 3)

FIG. 8C. MMLV-RT E302K nucleic acid sequence (SEQ ID NO: 4)

FIG. 8D. MMLV-RT E302R nucleic acid sequence (SEQ ID NO: 5)

FIG. 8E. MMLV-RT W313F nucleic acid sequence (SEQ ID NO: 6)

FIG. 8F. MMLV-RT L435G nucleic acid sequence (SEQ ID NO: 7)

FIG. 8G. MMLV-RT L435M nucleic acid sequence (SEQ ID NO: 8)

FIG. 8H. MMLV-RT N454K nucleic acid sequence (SEQ ID NO: 9)

FIG. 8I. MMLV-RT N454R nucleic acid sequence (SEQ ED NO: 10)

FIG. 8J. MMLV-RT D524N nucleic acid sequence (SEQ ID NO: 11)

FIG. 8K. MMLV-RT M651L nucleic acid sequence (SEQ ID NO: 12)

FIG. 8L. MMLV-RT E302R/E69K/W313F/L435G/N454K nucleic acid sequence (SEQID NO: 13)

FIG. 8M. MMLV-RT E302R/W313F/L435G/N454K nucleic acid sequence (SEQ IDNO: 14)

FIG. 8N. MMLV-RT E302R/W313F/L435G nucleic acid sequence (SEQ ID NO: 15)

FIG. 8O. MMLV-RT E302R/E69K/N454K nucleic acid sequence (SEQ ID NO: 16)

FIG. 8P. MMLV-RT E302R/W313F nucleic acid sequence (SEQ ID NO: 17)

FIG. 8Q. MMLV-RT E302R/E69K/W313F/L435G/N454K/D524N nucleic acidsequence (SEQ ID NO: 18)

FIG. 8R. WT-MMLV-RT protein sequence (SEQ ID NO: 19)

FIG. 8S. MMLV-RT E69K protein sequence (SEQ ID NO: 20)

FIG. 8T. MMLV-RT E302K protein sequence (SEQ ID NO: 21)

FIG. 8U. MMLV-RT E302R protein sequence (SEQ ID NO: 22)

FIG. 8V. MMLV-RT W313F protein sequence (SEQ ID NO: 23)

FIG. 8W. MMLV-RT L435G protein sequence (SEQ ID NO: 24)

FIG. 8X. MMLV-RT L435M protein sequence (SEQ ID NO: 25)

FIG. 8Y. MMLV-RT N454K protein sequence (SEQ ID NO: 26)

FIG. 8Z. MMLV-RT N454R protein sequence (SEQ ID NO: 27)

FIG. 8AA. MMLV-RT D524N protein sequence (SEQ ID NO: 28)

FIG. 8BB. MMLV-RT M651L protein sequence (SEQ ID NO: 29)

FIG. 8CC. MMLV-RT E302R/E69K/W313F/L435G/N454K protein sequence (SEQ IDNO: 30)

FIG. 8DD. MMLV-RT E302R/W313F/L435G/N454K protein sequence (SEQ ID NO:31)

FIG. 8EE. MMLV-RT E302R/W313F/L435G protein sequence (SEQ ID NO: 32)

FIG. 8FF. MMLV-RT E302R/E69K/N454K protein sequence (SEQ ID NO: 33)

FIG. 8GG. MMLV-RT E302R/W313F protein sequence (SEQ ID NO: 34)

FIG. 8HH. MMLV-RT E302R/E69K/W313F/L435G/N454K/D524N protein sequence(SEQ ID NO: 35)

FIG. 9A and FIG. 9B present the sequences of the epsilon subunit (c) ofE. coli DNA polymerase III holoenzyme (FIG. 9A) and the dnaQ gene (FIG.9B).

DETAILED DESCRIPTION

The invention relates to mutant reverse transcriptases (RTs). In oneembodiment the mutant RTs exhibit increased stability, for examplethermostability, as compared to a wild-type enzyme. The mutant RTs ofthe invention are useful for cDNA synthesis, cloning, production of cDNAlibraries or microarrays and RT-PCR.

I. REVERSE TRANSCRIPTASES

One common approach to the study of gene expression is the production ofcomplementary DNA (cDNA). Discovery of an RNA-dependent DNA polymerase,a so-called reverse transcriptase (RT), from a retrovirus has enabled areverse transcription reaction in which a cDNA is synthesized using anRNA as a template. As a result of identifying RT, methods for analyzingmRNA molecules have made rapid progress. The methods for analyzing mRNAmolecules using reverse transcriptase have now become indispensableexperimental methods for studying gene expression and function.Subsequently, these methods, which have been applied to cloning and PCRtechniques, have also become indispensable techniques in a wide varietyof fields including biology, medicine and agriculture.

The invention relates to a reverse transcriptase (RT) selected from thegroup consisting of: Moloney Murine Leukemia Virus (M-MLV) RT, HumanImmunodeficiency Virus (HIV) RT, Avian Sarcoma-Leukosis Virus (ASLV) RT,Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, AvianErythroblastosis Virus (AEV) Helper Virus MCAV RT, AvianMyelocytomatosis Virus MC29 Helper Virus MCAV RT, AvianReticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian SarcomaVirus UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus Y73 Helper VirusYAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis AssociatedVirus (MAV) RT.

Enzymes for use in the compositions, methods and kits of the presentinvention include any enzyme having reverse transcriptase activity. Suchenzymes include, but are not limited to, retroviral reversetranscriptase, retrotransposon reverse transcriptase, hepatitis Breverse transcriptase, cauliflower mosaic virus reverse transcriptase,E. coli DNA polymerase and klenow fragment, Tth DNA polymerase, Taq DNApolymerase (Saiki, R. K., et al., Science 239:487-491 (1988); U.S. Pat.Nos. 4,889,818 and 4,965,188), Tne DNA polymerase (WO 96/10640), Tma DNApolymerase (U.S. Pat. No. 5,374,553), C. Therm DNA polymerase fromCarboxydothermus hydrogenoformans (EP0921196A1, Roche, Pleasanton,Calif., Cat. No. 2016338), ThermoScript (Invitrogen, Carsbad, Calif.Cat. No. 11731-015) and mutants, fragments, variants or derivativesthereof. As will be understood by one of ordinary skill in the art,modified reverse transcriptases may be obtained by recombinant orgenetic engineering techniques that are routine and well-known in theart. Mutant reverse transcriptases can, for example, be obtained bymutating the gene or genes encoding the reverse transcriptase ofinterest by site-directed or random mutagenesis. Such mutations mayinclude point mutations, deletion mutations and insertional mutations.Preferably, one or more point mutations (e.g., substitution of one ormore amino acids with one or more different amino acids) are used toconstruct mutant reverse transcriptases of the invention. Fragments ofreverse transcriptases may be obtained by deletion mutation byrecombinant techniques that are routine and well-known in the art, or byenzymatic digestion of the reverse transcriptase(s) of interest usingany of a number of well-known proteolytic enzymes. Mutant DNApolymerases containing reverse transcriptase activity, for example, asdescribed in U.S. patent application Ser. No. 10/435,766, incorporatedby reference in its entirety, are also useful according to theinvention.

Polypeptides having reverse transcriptase activity that may beadvantageously used in the present methods include, but are not limitedto, Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, RousSarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus(AMV) reverse transcriptase, Rous-Associated Virus (RAV) reversetranscriptase, Myeloblastosis Associated Virus (MAV) reversetranscriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase,Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, retroviralreverse transcriptase, retrotransposon reverse transcriptase, hepatitisB reverse transcriptase, cauliflower mosaic virus reverse transcriptase,Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT®) DNApolymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™.Pyrococcus species GB-D DNA polymerase, Pyrococcus woesi (Pwo) DNApolymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacilluscaldophilus (Bca) DNA polymerase, Sulfoloblus acidocaldarius (Sac) DNApolymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermusflavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase,Thermus brockianus (DYNAZYME™) DNA polymerase, Methanobacteriumthermoautotrophicum (Mth) DNA polymerase, and mutants, variants andderivatives thereof. The invention also encompasses bacterial DNApolymerases comprising residual reverse transcriptase activity, such asTaq DNA polymerase (for a description see, for example, Shadilya et al.,2004 Extremophiles, 8:243).

Particularly preferred for use in the invention are the variants ofthese enzymes that are reduced in RNase H activity (i.e., RNaseH-enzymes). Preferably, the enzyme has less than 20%, more preferablyless than 15%, 10% or 5%, and most preferably less than 2%, of the RNaseH activity of a wildtype or “RNase H⁺” enzyme such as wildtype M-MLVreverse transcriptase. The RNase H activity of any enzyme may bedetermined by a variety of assays, such as those described, for example,in U.S. Pat. Nos. 5,244,797; 5,405,776; 5,668,005; and 6,063,608; inKotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988) and in Gerard,G. F., et al., FOCUS 14(5):91 (1992), the disclosures of all of whichare fully incorporated herein by reference.

Particularly preferred RNase H-reverse transcriptase enzymes for use inthe invention include, but are not limited to, M-MLV H-reversetranscriptase, RSV H-reverse transcriptase, AMV H-reverse transcriptase,RAV H-reverse transcriptase, MAV H-reverse transcriptase and HIVH-reverse transcriptase for example as previously described (see U.S.Pat. Nos. 5,244,797; 5,405,776; 5,668,005 and 6,063,608; and WO98/47912, the entirety of each is incorporated by reference). The RNaseH activity of any enzyme may be determined by a variety of assays, suchas those described, for example, in U.S. Pat. Nos. 5,244,797; 5,405,776;5,668,005 and 6,063,608; in Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988); and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), thedisclosures of all of which are fully incorporated herein by reference.It will be understood by one of ordinary skill, however, that any enzymecapable of producing a DNA molecule from a ribonucleic acid molecule(i.e., having reverse transcriptase activity) that is substantiallyreduced in RNase H activity may be equivalently used in thecompositions, methods and kits of the invention.

Polypeptides having reverse transcriptase activity for use in theinvention may be obtained commercially, for example, from Invitrogen,Inc. (Carlsbad, Calif.), Pharmacia (Piscataway, N.J.), Sigma (SaintLouis, Mo.) or Roche Molecular System (Pleasanton, Calif.).Alternatively, polypeptides having reverse transcriptase activity may beisolated from their natural viral or bacterial sources according tostandard procedures for isolating and purifying natural proteins thatare well-known to one of ordinary skill in the art (see, e.g., Houts, G.E., et al., J. Virol. 29:517 (1979)). In addition, the polypeptideshaving reverse transcriptase activity may be prepared by recombinant DNAtechniques that are familiar to one of ordinary skill in the art (see,e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis,D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376(1988)). The entire teaching of the above references is herebyincorporated by reference.

Enzymes that are reduced in RNase H activity may be obtained by methodsknown in the art, e.g., by mutating the RNase H domain within thereverse transcriptase of interest, preferably by one or more pointmutations, one or more deletion mutations, and/or one or more insertionmutations as described above, e.g., as described in U.S. Pat. No.6,063,608 hereby incorporated in its entirety by reference.

Two or more enzymes with reverse transcriptase activity may be used in asingle composition, e.g., the same reaction mixture. Enzymes used inthis fashion may have distinct reverse transcription pause sites withrespect to the template nucleic acid, as described in U.S. PatentApplication 2003/0198944A1, hereby incorporated in its entirety byreference.

The enzyme containing reverse transcriptase activity of the presentinvention may also include a mutant or modified reverse transcriptasewhere one or more amino acid changes have been made which renders theenzyme more faithful (higher fidelity) in nucleic acid synthesis, e.g.,as described in U.S. Patent Application 2003/0003452A1, herebyincorporated in its entirety by reference.

Epsilon Subunits

The invention provide for a reverse transcriptase of the invention incombination with a complex comprising the θ subunit of E. coli DNApolymerase III and the epsilon subunit of E. coli DNA polymerase III(e.g., see Hamdan et al., 2002, Biochemistry, 41:5266-5275). The θsubunit may also be used with any other mutant form of the epsilonsubunit, for example the epsilon 186 truncated version of the epsilonsubunit, to increase stability of the enzyme and/or to improve theaccuracy, specificity and or processivity of the reverse transcriptases.

In one embodiment of the invention, a mutant reverse transcriptase isprovided in combination with the θ epsilon subunit complex.Alternatively, a mutant reverse transcriptase is provided in combinationwith a complex comprising θ and a mutant form of the epsilon subunit,for example ε186

Denaturing Agents and Organic Solvents

The invention also provides for a reverse transcriptase in combinationwith a denaturing agent or organic solvent including but not limited toformamide and DMSO.

The invention also provides for a reverse transcriptase in combinationwith a PCR enhancing factor, for example, betaine.

II. GENETIC MODIFICATIONS Mutagenesis

The preferred method of preparing a mutant reverse transcriptase is bygenetic modification (e.g., by modifying the DNA sequence of a wild-typereverse transcriptase). A number of methods are known in the art thatpermit the random as well as targeted mutation of DNA sequences (see forexample, Ausubel et. al. Short Protocols in Molecular Biology (1995) 3rdEd. John Wiley & Sons, Inc.). In addition, there are a number ofcommercially available kits for site-directed mutagenesis, includingboth conventional and PCR-based methods. Examples include the GeneMorphRandom mutagenesis kit (Stratagene Catalog No. 600550 or 200550),EXSITE™ PCR-Based Site-directed Mutagenesis Kit available fromStratagene (Catalog No. 200502) and the QUIKCHANGE™ Site-directedmutagenesis Kit from Stratagene (Catalog No. 200518), and the CHAMELEON®double-stranded Site-directed mutagenesis kit, also from Stratagene(Catalog No. 200509).

In addition mutant reverse transcriptases may be generated byinsertional mutation or truncation (N-terminal, internal or C-terminal)according to methodology known to one skilled in the art.

Older methods of site-directed mutagenesis known in the art rely onsub-cloning of the sequence to be mutated into a vector, such as an M13bacteriophage vector, that allows the isolation of single-stranded DNAtemplate. In these methods, one anneals a mutagenic primer (i.e., aprimer capable of annealing to the site to be mutated but bearing one ormore mismatched nucleotides at the site to be mutated) to thesingle-stranded template and then polymerizes the complement of thetemplate starting from the 3′ end of the mutagenic primer. The resultingduplexes are then transformed into host bacteria and plaques arescreened for the desired mutation.

More recently, site-directed mutagenesis has employed PCR methodologies,which have the advantage of not requiring a single-stranded template. Inaddition, methods have been developed that do not require sub-cloning.Several issues must be considered when PCR-based site-directedmutagenesis is performed. First, in these methods it is desirable toreduce the number of PCR cycles to prevent expansion of undesiredmutations introduced by the polymerase. Second, a selection must beemployed in order to reduce the number of non-mutated parental moleculespersisting in the reaction. Third, an extended-length PCR method ispreferred in order to allow the use of a single PCR primer set. Andfourth, because of the non-template-dependent terminal extensionactivity of some thermostable polymerases it is often necessary toincorporate an end-polishing step into the procedure prior to blunt-endligation of the PCR-generated mutant product.

Non-limiting examples for the isolation of mutant reverse transcriptasesuseful according to the invention are described in detail in Examples 1and 2.

Methods of random mutagenesis, which will result in a panel of mutantsbearing one or more randomly situated mutations, exist in the art. Sucha panel of mutants may then be screened for those exhibiting the desiredproperties, for example, increased stability, relative to a wild-typereverse transcriptase. An example of a method for random mutagenesis isthe so-called “error-prone PCR method”. As the name implies, the methodamplifies a given sequence under conditions in which the DNA polymerasedoes not support high fidelity incorporation. Although the conditionsencouraging error-prone incorporation for different DNA polymerasesvary, one skilled in the art may determine such conditions for a givenenzyme. A key variable for many DNA polymerases in the fidelity ofamplification is, for example, the type and concentration of divalentmetal ion in the buffer. The use of manganese ion and/or variation ofthe magnesium or manganese ion concentration may therefore be applied toinfluence the error rate of the polymerase.

Genes for desired mutant reverse transcriptases generated by mutagenesismay be sequenced to identify the sites and number of mutations. Forthose mutants comprising more than one mutation, the effect of a givenmutation may be evaluated by introduction of the identified mutation tothe wild-type gene by site-directed mutagenesis in isolation from theother mutations borne by the particular mutant. Screening assays of thesingle mutant thus produced will then allow the determination of theeffect of that mutation alone.

The amino acid and DNA coding sequence of wild-type MMLV-reversetranscriptase are shown in FIG. 8. Non-limiting detailed procedures forpreparing a mutant MMLV-reverse transcriptase useful according to theinvention are provided in Examples 1 and 2.

A person of average skill in the art having the benefit of thisdisclosure will recognize that mutant reverse transcriptases polymerasesderived from other reverse transcriptases, including but not limited toMoloney Murine Leukemia Virus (M-MLV); Human Immunodeficiency Virus(HIV) reverse transcriptase and avian Sarcoma-Leukosis Virus (ASLV)reverse transcriptase, which includes but is not limited to Rous SarcomaVirus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV)reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper VirusMCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 HelperVirus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus(REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma VirusUR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV)reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reversetranscriptase may be suitably used in the subject compositions.

The enzyme of the subject composition may comprise reversetranscriptases that have not yet been isolated.

A method employing the addition of peptide tails with random sequencesto the C-terminus of Bacillus stearothermophilus Catalase I, in anattempt to increase enzyme thermostability has been described (Matsuuraet al., 1999 Nature Biotechnology 17:58). The invention contemplatesmutant reverse transcriptases comprising a C-terminal extension.

As used herein, a “C-terminal extension” refers to a peptide tail ofrandom sequence. A C-terminal extension is preferably from 1 to 500amino acids, more preferably from 1 to 100 amino acids, and mostpreferably from 2 to 50 amino acids.

III. METHODS OF EVALUATING MUTANTS FOR INCREASED THERMOSTABILITY

Random or site-directed mutants generated as known in the art or asdescribed herein and expressed in bacteria may be screened for RTactivity and increased stability of RT activity by several differentassays. Preferably, an RT enzyme is screened in an RT thermostabilityscreen as described in Example 3, hereinbelow.

IV. EXPRESSION OF WILD-TYPE OR MUTANT ENZYMES ACCORDING TO THE INVENTION

Methods known in the art may be applied to express and isolate themutated forms of reverse transcriptase according to the invention. Themethods described here can be also applied for the expression ofwild-type enzymes useful in the invention. Many bacterial

expression vectors contain sequence elements or combinations of sequenceelements allowing high level inducible expression of the protein encodedby a foreign sequence. For example, bacteria expressing an integratedinducible form of the T7 RNA polymerase gene may be transformed with anexpression vector bearing a mutated DNA polymerase gene linked to the T7promoter. Induction of the T7 RNA polymerase by addition of anappropriate inducer, for example, isopropyl-β-D-thiogalactopyranoside(IPTG) for a lac-inducible promoter, induces the high level expressionof the mutated gene from the T7 promoter.

Appropriate host strains of bacteria may be selected from thoseavailable in the art by one of skill in the art. As a non-limitingexample, E. coli strain BL-21 is commonly used for expression ofexogenous proteins since it is protease deficient relative to otherstrains of E. coli. BL-21 strains bearing an inducible T7 RNA polymerasegene include WJ56 and ER2566 (Gardner & Jack, 1999, supra). Forsituations in which codon usage for the particular reverse transcriptasegene differs from that normally seen in E. coli genes, there are strainsof BL-21 that are modified to carry tRNA genes encoding tRNAs with rareranticodons (for example, argU, ileY, leuW, and proL tRNA genes),allowing high efficiency expression of cloned protein genes, forexample, cloned archaeal enzyme genes (several BL21-CODON PLUS™ cellstrains carrying rare-codon tRNAs are available from Stratagene, forexample).

V. APPLICATIONS OF THE SUBJECT INVENTION

cDNA Synthesis

In accordance with the invention, cDNA molecules (single-stranded ordouble-stranded) may be prepared from a variety of nucleic acid templatemolecules. Preferred nucleic acid molecules for use in the presentinvention include single-stranded or double-stranded DNA and RNAmolecules, as well as double-stranded DNA:RNA hybrids. More preferrednucleic acid molecules include messenger RNA (mRNA), transfer RNA (tRNA)and ribosomal RNA (rRNA) molecules, although mRNA molecules are thepreferred template according to the invention.

The invention provides compositions and methods for cDNA synthesis withincreased specificity and accuracy. The present invention providescompositions and methods for high fidelity cDNA synthesis. The subjectcompositions and methods may also increase the efficiency of the reversetranscription as well as the length of the cDNA synthesized. As aresult, the fidelity, efficiency, and yield of subsequent manipulationsof the synthesized cDNA (e.g., amplification, sequencing, cloning, etc.)are also increased. The nucleic acid molecules that are used to preparecDNA molecules according to the methods of the present invention may beprepared synthetically according to standard organic chemical synthesismethods that will be familiar to one of ordinary skill. More preferably,the nucleic acid molecules may be obtained from natural sources, such asa variety of cells, tissues, organs or organisms. Cells that may be usedas sources of nucleic acid molecules may be prokaryotic (bacterialcells, including but not limited to those of species of the generaEscherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Xanthomonas and Streptomyces) or eukaryotic(including fungi (especially yeasts), plants, protozoans and otherparasites, and animals including insects (particularly Drosophila spp.cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly human cells)).

Mammalian somatic cells that may be used as sources of nucleic acidsinclude blood cells (reticulocytes and leukocytes), endothelial cells,epithelial cells, neuronal cells (from the central or peripheral nervoussystems), muscle cells (including myocytes and myoblasts from skeletal,smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes)may also be used as sources of nucleic acids for use in the invention,as may the progenitors, precursors and stem cells that give rise to theabove somatic and germ cells. Also suitable for use as nucleic acidsources are mammalian tissues or organs such as those derived frombrain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous,skin, genitourinary, circulatory, lymphoid, gastrointestinal andconnective tissue sources, as well as those derived from a mammalian(including human) embryo or fetus.

Any of the above prokaryotic or eukaryotic cells, tissues and organs maybe normal, diseased, transformed, established, progenitors, precursors,fetal or embryonic. Diseased cells may, for example, include thoseinvolved in infectious diseases (caused by bacteria, fungi or yeast,viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) orparasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, muscular dystrophy ormultiple sclerosis) or in cancerous processes. Transformed orestablished animal cell lines may include, for example, COS cells, CHOcells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293cells, L929 cells, F9 cells, and the like. Other cells, cell lines,tissues, organs and organisms suitable as sources of nucleic acids foruse in the present invention will be apparent to one of ordinary skillin the art.

Once the starting cells, tissues, organs or other samples are obtained,nucleic acid molecules (such as mRNA) may be isolated therefrom bymethods that are well-known in the art (See, e.g., Maniatis, T., et al.,Cell 15:687-701 (1978); Okayama, H., and Berg, P., Mol. Cell. Biol.2:161-170 (1982); Gubler, U., and Hoffman, B. J., Gene 25:263-269(1983)). The nucleic acid molecules thus isolated may then be used toprepare cDNA molecules and cDNA libraries in accordance with the presentinvention.

In the practice of the invention, cDNA molecules or cDNA libraries maybe produced by mixing one or more nucleic acid molecules obtained asdescribed above, which is preferably one or more mRNA molecules such asa population of mRNA molecules, with the composition of the invention,under conditions favoring the reverse transcription of the nucleic acidmolecule by the action of the enzymes of the compositions to form a cDNAmolecule (single-stranded or double-stranded). Thus, the method of theinvention comprises (a) mixing one or more nucleic acid templates(preferably one or more RNA or mRNA templates, such as a population ofmRNA molecules) with a mutant RT of the invention and (b) incubating themixture under conditions sufficient to permit cDNA synthesis, e.g., toall or a portion of the one or more templates.

The compositions of the present invention may be used in conjunctionwith methods of cDNA synthesis such as those described in the Examplesbelow, or others that are well-known in the art (see, e.g., Gubler, U.,and Hoffman, B. J., Gene 25:263-269 (1983); Krug, M. S., and Berger, S.L., Meth. Enzymol. 152:316-325 (1987); Sambrook, J., et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, pp. 8.60-8.63 (1989)), to produce cDNAmolecules or libraries.

The invention is directed to such methods which further produce a firststrand and a second strand cDNA, as known in the art. According to theinvention, the first and second strand cDNAs produced by the methods mayform a double stranded DNA molecule which may be a full length cDNAmolecule.

Other methods of cDNA synthesis which may advantageously use the presentinvention will be readily apparent to one of ordinary skill in the art.

Subsequent Manipulation of Synthesized cDNA

Having obtained cDNA molecules or libraries according to the presentmethods, these cDNAs may be isolated or the reaction mixture containingthe cDNAs may be directly used for further analysis or manipulation.Detailed methodologies for purification of cDNAs are taught in theGENETRAPPER™ manual (Invitrogen, Inc. Carlsbad, Calif.), which isincorporated herein by reference in its entirety, although alternativestandard techniques of cDNA isolation such as those described in theExamples below or others that are known in the art (see, e.g., Sambrook,J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 8.60-8.63 (1989))may also be used.

In other aspects of the invention, the invention may be used in methodsfor amplifying nucleic acid molecules. Nucleic acid amplificationmethods according to this aspect of the invention may be one-step (e.g.,one-step RT-PCR) or two-step (e.g., two-step RT-PCR) reactions.According to the invention, one-step RT-PCR type reactions may beaccomplished in one tube thereby lowering the possibility ofcontamination. Such one-step reactions comprise (a) mixing a nucleicacid template (e.g., mRNA) with an enzyme of the present invention and(b) incubating the mixture under conditions sufficient to permitamplification. Two-step RT-PCR reactions may be accomplished in twoseparate steps. Such a method comprises (a) mixing a nucleic acidtemplate (e.g., mRNA) with an enzyme of the present invention, (b)incubating the mixture under conditions sufficient to permit cDNAsynthesis, (c) mixing the reaction mixture in (b) with one or more DNApolymerases and (d) incubating the mixture of step (c) under conditionssufficient to permit amplification. For amplification of long nucleicacid molecules (i.e., greater than about 3-5 Kb in length), acombination of DNA polymerases may be used, such as one DNA polymerasehaving 3′-5′ exonuclease activity and another DNA polymerase beingreduced in 3′-5′ exonuclease activity.

Amplification methods which may be used in accordance with the presentinvention include PCR (e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202),Strand Displacement Amplification (SDA; e.g., U.S. Pat. No. 5,455,166;EP 0 684 315), and Nucleic Acid Sequence-Based Amplification (NASBA;e.g., U.S. Pat. No. 5,409,818; EP 0 329 822). In a particularlypreferred aspects, the invention may be used in methods of amplifyingnucleic acid molecule comprising one or more polymerase chain reactions(PCRs), such as any of the PCR-based methods described above. Allreferences are entirely incorporated by reference.

Various specific PCR amplification applications are available in the art(for reviews, see for example, Erlich, 1999, Rev Immunogenet., 1:127-34;Prediger 2001, Methods Mol. Biol. 160:49-63; Jurecic et al., 2000, Curr.Opin. Microbiol. 3:316-21; Triglia, 2000, Methods Mol. Biol. 130:79-83;MaClelland et al., 1994, PCR Methods Appl. 4:S66-81; Abramson and Myers,1993, Current Opinion in Biotechnology 4:41-47; each of which isincorporated herein by references).

The subject invention can be used in PCR applications including, but notlimited to, i) hot-start PCR which reduces non-specific amplification;ii) touch-down PCR which starts at high annealing temperature, thendecreases annealing temperature in steps to reduce non-specific PCRproduct; iii) nested PCR which synthesizes more reliable product usingan outer set of primers and an inner set of primers; iv) inverse PCR foramplification of regions flanking a known sequence. In this method, DNAis digested, the desired fragment is circularized by ligation, then PCRusing primer complementary to the known sequence extending outwards; v)AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA). Thesemethods create genomic fingerprints from species with little-knowntarget sequences by amplifying using arbitrary oligonucleotides; vi)RT-PCR which uses RNA-directed DNA polymerase (e.g., reversetranscriptase) to synthesize cDNAs which is then used for PCR. Thismethod is extremely sensitive for detecting the expression of a specificsequence in a tissue or cells. It may also be used to quantify mRNAtranscripts; vii) RACE (rapid amplification of cDNA ends). This is usedwhere information about DNA/protein sequence is limited. The methodamplifies 3′ or 5′ ends of cDNAs generating fragments of cDNA with onlyone specific primer each (plus one adaptor primer). Overlapping RACEproducts can then be combined to produce full length cDNA; viii) DD-PCR(differential display PCR) which is used to identify differentiallyexpressed genes in different tissues. The first step in DD-PCR involvesRT-PCR, then amplification is performed using short, intentionallynonspecific primers; ix) Multiplex-PCR in which two or more uniquetargets of DNA sequences in the same specimen are amplifiedsimultaneously. One DNA sequence can be used as control to verify thequality of PCR; x) Q/C-PCR (Quantitative comparative) which uses aninternal control DNA sequence (but of different size) which competeswith the target DNA (competitive PCR) for the same set of primers; xi)Recusive PCR which is used to synthesize genes. Oligonucleotides used inthis method are complementary to stretches of a gene (>80 bases),alternately to the sense and to the antisense strands with endsoverlapping (˜20 bases); xii) Asymmetric PCR; xiii) In Situ PCR; xiv)Site-directed PCR Mutagenesis.

It should be understood that this invention is not limited to anyparticular amplification system. As other systems are developed, thosesystems may benefit by practice of this invention.

The primer used for synthesizing a cDNA from an RNA as a template in thepresent invention is not limited to a specific one as long as it is anoligonucleotide that has a nucleotide sequence complementary to that ofthe template RNA and that can anneal to the template RNA under reactionconditions used. The primer may be an oligonucleotide such as anoligo(dT) or an oligonucleotide having a random sequence (a randomprimer) or a gene-specific primer.

The nucleic acid molecules (e.g., synthesized cDNA or amplified product)or cDNA libraries prepared by the methods of the present invention maybe further characterized, for example by cloning and sequencing (i.e.,determining the nucleotide sequence of the nucleic acid molecule), bythe sequencing methods of the invention or by others that are standardin the art (see, e.g., U.S. Pat. Nos. 4,962,022 and 5,498,523, which aredirected to methods of DNA sequencing). Alternatively, these nucleicacid molecules may be used for the manufacture of various materials inindustrial processes, such as hybridization probes by methods that arewell-known in the art. Production of hybridization probes from cDNAswill, for example, provide the ability for those in the medical field toexamine a patient's cells or tissues for the presence of a particulargenetic marker such as a marker of cancer, of an infectious or geneticdisease, or a marker of embryonic development. Furthermore, suchhybridization probes can be used to isolate DNA fragments from genomicDNA or cDNA libraries prepared from a different cell, tissue or organismfor further characterization.

It is understood that the amplified product produced using the subjectenzyme can be cloned by any method known in the art. In one embodiment,the invention provides a composition which allows direct cloning of PCRamplified product.

The most common method for cloning PCR products involves incorporationof flanking restriction sites onto the ends of primer molecules. The PCRcycling is carried out and the amplified DNA is then purified,restricted with an appropriate endonuclease(s) and ligated to acompatible vector preparation.

A method for directly cloning PCR products eliminates the need forpreparing primers having restriction recognition sequences and it wouldeliminate the need for a restriction step to prepare the PCR product forcloning. Additionally, such method would preferably allow cloning PCRproducts directly without an intervening purification step.

U.S. Pat. Nos. 5,827,657 and 5,487,993 (hereby incorporated by theirentirety) disclose methods for direct cloning of PCR products using aDNA polymerase which takes advantage of the single 3′-deoxy-adenosinemonophosphate (dAMP) residues attached to the 3′ termini of PCRgenerated nucleic acids. Vectors are prepared with recognition sequencesthat afford single 3′-terminal deoxy-thymidine monophosphate (dTMP)residues upon reaction with a suitable restriction enzyme. Thus, PCRgenerated copies of genes can be directly cloned into the vectorswithout need for preparing primers having suitable restriction sitestherein.

Taq DNA polymerase exhibits terminal transferase activity that adds asingle dATP to the 3′ ends of PCR products in the absence of template.This activity is the basis for the TA cloning method in which PCRproducts amplified with Taq are directly ligated into vectors containingsingle 3′dT overhangs. Pfu DNA polymerase, on the other hand, lacksterminal transferase activity, and thus produces blunt-ended PCRproducts that are efficiently cloned into blunt-ended vectors.

In one embodiment, the invention provides for a PCR product, generatedin the presence of a mutant DNA polymerase with reduced uracil detectionactivity, that is subsequently incubated with Taq DNA polymerase in thepresence of dATP at 72° C. for 15-30 minutes. Addition of 3′-dAMP to theends of the amplified DNA product then permits cloning into TA cloningvectors according to methods that are well known to a person skilled inthe art.

The nucleic acid molecules (e.g., synthesized cDNA or amplified product)of the present invention may also be used to prepare compositions foruse in recombinant DNA methodologies. Accordingly, the present inventionrelates to recombinant vectors which comprise the cDNA or amplifiednucleic acid molecules of the present invention, to host cells which aregenetically engineered with the recombinant vectors, to methods for theproduction of a recombinant polypeptide using these vectors and hostcells, and to recombinant polypeptides produced using these methods.

Recombinant vectors may be produced according to this aspect of theinvention by inserting, using methods that are well-known in the art,one or more of the cDNA molecules or amplified nucleic acid moleculesprepared according to the present methods into a vector. The vector usedin this aspect of the invention may be, for example, a phage or aplasmid, and is preferably a plasmid. Preferred are vectors comprisingcis-acting control regions to the nucleic acid encoding the polypeptideof interest. Appropriate trans-acting factors may be supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression (and are therefore termed “expression vectors”),which may be inducible and/or cell type-specific. Particularly preferredamong such vectors are those inducible by environmental factors that areeasy to manipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids, and will preferablyinclude at least one selectable marker such as a tetracycline orampicillin resistance gene for culturing in a bacterial host cell. Priorto insertion into such an expression vector, the cDNA or amplifiednucleic acid molecules of the invention should be operatively linked toan appropriate promoter, such as the phage lambda P_(L) promoter, the E.coli lac, trp and tac promoters. Other suitable promoters will be knownto the skilled artisan.

Among vectors preferred for use in the present invention include pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; pcDNA3 available from Invitrogen; pGEX, pTrxfus,pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 available fromPharmacia; and pSPORT1, pSPORT2 and pSV.multidot.SPORT1, available fromLife Technologies, Inc. Other suitable vectors will be readily apparentto the skilled artisan.

The invention also provides methods of producing a recombinant host cellcomprising the cDNA molecules, amplified nucleic acid molecules orrecombinant vectors of the invention, as well as host cells produced bysuch methods. Representative host cells (prokaryotic or eukaryotic) thatmay be produced according to the invention include, but are not limitedto, bacterial cells, yeast cells, plant cells and animal cells.Preferred bacterial host cells include Escherichia coli cells (mostparticularly E. coli strains DH1OB and Stb12, which are availablecommercially (Life Technologies, Inc; Rockville, Md.)), Bacillussubtilis cells, Bacillus megaterium cells, Streptomyces spp. cells,Erwinia spp. cells, Klebsiella spp. cells and Salmonella typhimuriumcells. Preferred animal host cells include insect cells (mostparticularly Spodoptera frugiperda 5.19 and Sf21 cells and TrichoplusaHigh-Five cells) and mammalian cells (most particularly CHO, COS, VERO,BHK and human cells). Such host cells may be prepared by well-knowntransformation, electroporation or transfection techniques that will befamiliar to one of ordinary skill in the art.

In addition, the invention provides methods for producing a recombinantpolypeptide, and polypeptides produced by these methods. According tothis aspect of the invention, a recombinant polypeptide may be producedby culturing any of the above recombinant host cells under conditionsfavoring production of a polypeptide therefrom, and isolation of thepolypeptide. Methods for culturing recombinant host cells, and forproduction and isolation of polypeptides therefrom, are well-known toone of ordinary skill in the art.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

VI. Kits

The present compositions may be assembled into kits for use in reversetranscription, cloning or amplification of a nucleic acid molecule. Kitsaccording to this aspect of the invention comprise a carrier means, suchas a box, carton, tube or the like, having in close confinement thereinone or more container means, such as vials, tubes, ampules, bottles andthe like. The kits of the invention may also comprise (in the same orseparate containers) one or more reverse transcriptases, a suitablebuffer, one or more nucleotides and/or one or more primers or any otherreagents described for compositions of the present invention.

The kit of the present invention may include reagents facilitating thesubsequent manipulation of cDNA synthesized as known in the art.

VII. EXAMPLES Example 1 Generating RNase H Minus MMLV-RT Point MutantLibrary for Thermostability Screen

RNase H minus MMLV-RT (D524N) gene (2 kb) was mutagenized using theGeneMorph Random Mutagenesis Kit (Stratagene Catalog #200550 or 600550)and primers pSTRAT-F and pSTRAT-R (Table 2) according to themanufacturer's recommendations. Mutated PCR products “Mega primers” wereused to replace the wild type RNase H minus MMLV-RT gene using theQuikChange Site-directed Mutagenesis Kit (Stratagene Catalog #200518)according to the manufacturer's recommendations. The resulting plasmidswere cloned into XL-10 Gold competent cells (Stratagene Catalog#200317). The library size was 5×10⁴ (containing 1-6 mutations/kb). DNAwas extracted from the entire library using StrataPrep Plasmid MiniprepKit (Stratagene Catalog #400761). A portion of the DNA was thentransformed into BL21-DE3-RIL cells (Stratagene Catalog #230240) togenerate a library with a size of 5×10⁴.

Results: The clones in this library contained 1-6 mutations/kb.

Example 2 Generating RNase H Minus MMLV-RT Random C-Terminal ExtensionLibrary for Thermostability Screen

Primers RTSSC12AXhoI and RTSSEI-vecF (Table 2) were used to amplifyRNase H minus MMLV-RT gene using Herculase DNA polymerase (StratageneCatalog #600260). The PCR products were then digested with EcoRI andXhol and cloned into pCal-n-FLAG (Stratagene Catalog #214311) that ismissing the Calmodulin binding unit and the FLAG sequence. The resultingC-terminal extension library was cloned into XL-10 Gold competent cells(Stratagene Catalog #200317). DNA was extracted from the entire libraryusing StrataPrep Plasmid Miniprep Kit (Stratagene Catalog #400761) andtransformed into BL21-DE3-RIL cells (Stratagene Catalog #230240).

Results:

The library size was 10⁴. From 17 clones sequenced, 12 had 7-14 aminoacid additions, 2 had 1-2 amino acid additions, 1 had 18 amino acidadditions, 1 had 30 amino acid additions, and one had no additions.

Example 3 RT Thermostability Screen Assay

Mutant colonies from the BL21-DE3-RIL libraries (both point mutant andC-terminal extension libraries) were inoculated into 120 μI LB mediacontaining 100 μg/ml Ampicillin and 35 μg/ml Chloramphenicol (Costar 96well plate (29444-102)) and grown over night at 37° C. 10 μI of thesecultures were inoculated into 110 μl LB media containing 100 μg/mlAmpicillin, 35 μg/ml Chloramphenicol, and 1 mM IPTG (Costar 96 wellplate (29444-102)) and grown over night. Cells were lysed using 30 μllysis buffer (125 mM Tris pH 8, 4.5% glucose, 50 mM EDTA, 2.5% Triton, 5mg/ml lysozyme, and 50 mM DTT). 10 μl of lysates were used in a 50 μlassay containing 50 mM Tris pH 8.3, 75 ml KCl, 8 mM MgCl₂, 2 μgpoly(rC), 0.5 μg oligo(dG), 10 mM DTT, 50 mM dGTP, and 0.5 μCi α³³pdGTP.Reactions were incubated at 42° C. or 55° C. for 60 minutes (FIG. 1). 4μl of these reactions were spotted on DE-81 filters, and dried. Thefilters were then washed 5 times with 2×SSC and dried. The filters werethen exposed to Kodak BioMax MR-1 films (VWR IB8941114)) over night.

Results:

3400 clones from the point mutation library were screened using thethermostability assay described above. The mutants that showed higheractivity at 55° C. compared to the WT enzyme (FIG. 1) were selected andre-screened using the same RT activity assay three more times. The bestmutants were selected, sequenced, and His-tag purified (as in example6). Mutations E69K, L435M, N454K, and M651L were discovered and their RTactivity at 52° C./42° C. (as in example 4) were compared to the WTenzyme (FIG. 2). All His-tagged purified mutants showed higher activityat 52° C./42° C. compared to the WT enzyme.

4000 clones from the C-terminal extension library were also screenedusing the thermostability assay described above. The mutants that showedhigher activity at 55° C. compared to the WT enzyme were selected andre-screened using the same RT activity assay three more times. The bestmutants were selected, sequenced, and His-tag purified (as in example6). Multiple peptide tails increased the activity of RT at 52° C./42° C.(assayed as in example 4) compared to the WT enzyme (FIG. 3).

Example 4 RT Activity Assay

The RNA dependent DNA polymerization assays for His-tagged purified WTand mutants were performed as follows. ˜5 units of each enzyme(equivalent amount of protein on a SDS-PAGE gel) were used in a 50 μlassay containing 50 mM Tris pH 8.3, 75 mM KCl, 8 mM MgCl₂, 2 μgpoly(rC), 0.5 μg oligo(dG), 10 mM DTT, 50 mM dGTP, and 0.5 μCi α³³pdGTP.Reactions were incubated at 42° C. or 52° C. for 30 minutes. 5 μl ofthese reactions were spotted on DE-81 filters, and dried. The filterswere then washed 5 times with 2×SSC, followed by a brief wash with 100%ethanol. The filters were then dried. Incorporated radioactivity wasmeasured by scintillation counting. Reactions that lacked enzyme wereset up along with sample incubations to determine “total cpms” (omitfilter wash steps) and “minimum cpms” (wash filters as above). Minimumcpms were subtracted from sample cpms to determine “corrected cpms”.

Example 5 Saturation Mutagenesis at Putative “Thermostability” Residuesto Identify Best Mutation at Each Site Independently

Saturation mutagenesis was performed using QuikChange Site-directedMutagenesis Kit (Stratagene Catalog #200518) and primers containingdegenerate site (NNG/T) at E69, E302, F303, G305, W313, L435, N454, M651(Table 2) according to the manufacturer's recommendations. 200 clonesfrom every library were screened (as in example 3). The mutants with thehighest activity at 55° C. were selected, and sequenced.

Results:

The following mutations show the highest activity at 55° C.:

E69K, E302K, E302R, W313F, L435M, L435G, N454K, N454R, M651L

Example 6 Combination of Thermostable Mutations

The QuikChange Multi Site-directed Mutagenesis Kit (Stratagene Catalog#200514) with four primers (Table 2) was used to introduce the mutationsE69K, W313F, L435G, and N454K into an RNase H minus MMLV-RT gene thatalready contained the E302R mutation.

Ten clones were sequenced.

Results: The following combinations were obtained:

Clone 1: E302R/E69K/W313F/L435G/ RKFGK N454K (SEQ ID NO: 36) Clone 2:E302R/W313F/L435G/N454K RFGK (SEQ ID NO: 37) Clone 3: E302R/W313F/L435GRFG Clone 4: E302R/E69K/N454K RKK Clone 5: E302R/W313F RF

Activity assays (as in example 3—using DE-81 filters andpoly(rC):oligo(dG)₁₈) (SEQ ID NO:62) were performed at 42° C. and 57° C.and the results (FIG. 4) indicate higher activity at 57° C. for clonescontaining single or multiple mutations as compared to the wild typeenzyme.

Example 7 Activity Assay Using Poly(A) RNA Ladder

Full length cDNA profiling was performed for WT RT versus RKFGK (SEQ IDNO:36) mutant RT (His-tagged proteins) using a poly(A)-tailed RNA ladder(Ambion #7150). Reactions contained 2 μg RNA ladder, 0.5 μs oligo(dT)₁₈(SEQ ID NO:63), 3.2 mM dNTPs and ˜100 units of enzyme (equivalentprotein amount on a SDS-PAGE gel) in 1× StrataScript buffer containing 3or 6 mM Mg²⁺. Reactions were incubated at 42° C., 50° C., and 52° C. for60 minutes, run on a 1% alkaline agarose gel and stained with SYBR Gold.

Results:

RKFGK (SEQ ID NO: 36) mutant RT generates longer cDNA ladders at highertemperature (52° C.) compared to the WT enzyme (FIG. 5).

Example 8 Purification and Thermostability Comparison of FinalConstructs

Three His tagged constructs including RNase H minus MMLV-RT (D524N),RNase H minus MMLV-RT (D524N, E302R, E69K, W313F, L435G, N454K), andRNase H minus MMLV-RT (D524N, E302R, E69K, W313F, L435G, N454K) plus theC-terminal extension (RDRNKNNDRRKAKENE) (SEQ ID NO: 1) were expressedand purified according to the QIAexpressionist (Qiagen). An RT activityassay using Poly(rC):poly(dG) was performed similar to as in Example 4.

Results: The RNase H minus MMLV-RT (D524N, E302R, E69K, W313F, L435G,N454K) with the C-terminal extension (RDRNKNNDRRKAKENE) (SEQ ID NO: 1)shows the highest activity at 55° C. and 60° C. (FIG. 6).

Example 9 Half Life Determination

Half-lives of mutant reverse transcriptase enzymes of the invention weredetermined as follows.

Three non-His tagged constructs including RNase H minus MMLV-RT (D524N)

(FIG. 7A, plot 1), RNase H minus MMLV-RT (D524N, E302R, E69K, W313F,L435G, N454K) (FIG. 7B, plot 2), and RNase H minus MMLV-RT(D524N, E302R,E69K, W313F, L435G, N454K) plus the C-terminal extension(RDRNKNNDRRKAKENE) (SEQ ID NO: 1) (FIG. 7C, plot 3) were assayed asfollows. Mixtures containing 0.5 pmol of each enzyme in the presence of2 μg poly(rC), 0.5 μg oligo(dG)₁₈ (SEQ ID NO:62) were incubated at 55°C. for various times as indicated in the plots. Incubation was stoppedby placing the tubes on ice. An aliquot was assayed for residualactivity in 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 50 mMdGTP, and 0.5 μCi α³³pdGTP. Reactions were incubated at 42° C. for 30minutes. 5 μl of these reactions were spotted on DE-81 filters, anddried. The filters were then washed 5 times with 2×SSC, followed by abrief wash with 100% ethanol. The filters were then dried. Incorporatedradioactivity was measured by scintillation counting. Reactions thatlacked enzyme were set up along with sample incubations to determine“total cpms” (omit filter wash steps) and “minimum cpms” (wash filtersas above). Minimum cpms were subtracted from sample cpms to determine“corrected cpms”.

Results:

The half life of RNase H minus MMLV-RT (D524N) (FIG. 7A, plot 1) is <5minutes where the half life of RNase H minus MMLV-RT (D524N, E302R,E69K, W313F, L435G, N454K) (FIG. 7B, plot 2) is >30 minutes, and thehalf life of RNase H minus MMLV-RT (D524N, E302R, E69K, W313F, L435G,N454K) plus the C-terminal extension (RDRNKNNDRRKAKENE) (SEQ ID NO: 1)(FIG. 7C, plot 3) if ˜30 minutes at 55° C.

TABLE 1 Primer sequences pSTRAT-F: 5′-ACCCTAAATATAGAAGATGAGCATCG(SEQ ID NO: 38) pSTRAT-R: 5′-GAGGAGGGTAGAGGTGTCTGGAGTC (SEQ ID NO: 39)RTSSC12AXhoI: 5′-CTTGGCCAAGGATCCGCTCGAGCTACTTACTTANNNNNNNNNNNNNNNNNNNNNGAGGAGGGTAGAGGTGT C (SEQ ID NO. 40) RTSSEI-vecF:5′-AGCGGATAACAATTCCCCTCTAGAATTCGA (SEQ ID NO: 41) pE69X-F:5′-CCCATGTCACAANNKGCCAGACTGGG K = G or T (SEQ ID NO: 42) pE302X-F:5′-GACAACTAAGGNNKTTCCTAGGGACG (SEQ ID NO: 43) pF303X-F:5′-CAACTAAGGGAGNNKCTAGGGACGGC (SEQ ID NO: 44) pG305X-F:5′-GGAGTTCCTANNKACGGCAGGCTTC (SEQ ID NO: 45) pW313X-F:5′-TCTGTCGCCTCNNKATCCCTGGGTTTG (SEQ ED NO: 46) pL435X-F:5′-CCACTAGTCATTNNKGCCCCCCATGCAG (SEQ ID NO: 47) pN454X-F:5′-GCTGGCTTTCCNNKGCCCGGATGACTC (SEQ ID NO: 48) pM651X-F5′-GAGGCAACCGGNNKGCTGACCAAGCG (SEQ ID NO: 49) pE69X-R:5′-CCCAGTCTGGCMNNTTGTGACATGGG M = A or C (SEQ ID NO: 50) pE302X-R:5′-CGTCCCTAGGAAMNNCCTTAGTTGTC (SEQ ID NO: 51) pF303X-R:5′-GCCGTCCCTAGMNNCTCCCTTAGTTG (SEQ ID NO: 52) pG305X-R:5′-GAAGCCTGCCGTMNNTAGGAACTCC (SEQ ID NO: 53) pW313X-R:5 CAAACCCAGGGATMNNGAGGCGACAGA (SEQ ID NO: 54) pL435X-R:5′-CTGCATGGGGGGCMNNAATGACTAGTGG (SEQ ID NO: 55) pN454X-R:5′-GAGTCATCCGGGCMNNGGAAAGCCAGC (SEQ ID NO: 56) pM651X-R:5 CGCTTGGTCAGCMNNCCGGTTGCCTC (SEQ ID NO: 57) pE69K:5′-TACCCCATGTCACAAAAAGCCAGACTGGGGATCA AG (SEQ ID NO: 58) pW313F:5′-GGCTTCTGTCGCCTCTTTATCCCTGGGTTTGC (SEQ ID NO: 59) pL435G:5′-CAGCCACTAGTCATTGGGGCCCCCCATGCAGTAG (SEQ ID NO: 60) pN454K:5′-GACCGCTGGCTTTCCAAGGCCCGGATGACTCAC (SEQ ID NO: 61)

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1-41. (canceled)
 42. An isolated mutant MMLV reverse transcriptasehaving reverse transcriptase activity comprising the sequence of SEQ IDNO:19 with the exception of mutations at at least three of the followingamino acid positions: E69, E302, W313, L435, N454, and M651.
 43. Theisolated mutant MMLV reverse transcriptase of claim 42, wherein thereverse transcriptase comprises at least one of the following mutations:a glutamic acid to lysine mutation at position E69, a glutamic acid tolysine mutation at position E302, a glutamic acid to arginine mutationat position E302, a tryptophan to phenylalanine mutation at positionW313, a leucine to glycine mutation at position L435, a leucine tomethionine mutation at position L435, an asparagine to lysine mutationat position N454, an asparagine to arginine mutation at position N454,and a methionine to leucine mutation at position M651.
 44. The isolatedmutant MMLV reverse transcriptase of claim 42, wherein the reversetranscriptase comprises a combination of mutations selected from thefollowing groups of combinations of mutations:E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; andE69K/E302R/W313F/L435G/N454K/D524N.
 45. The isolated mutant MMLV reversetranscriptase of claim 42, further comprising a C-terminal extension.46. The isolated mutant MMLV reverse transcriptase of claim 45, whereinsaid C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1).
 47. Theisolated mutant MMLV reverse transcriptase of claim 42, wherein saidreverse transcriptase lacks RNase H activity.
 48. The isolated mutantMMLV reverse transcriptase of claim 42, wherein the reversetranscriptase has at least one of the following characteristics:increased stability, increased accuracy, increased processivity, andincreased specificity.
 49. A composition comprising the isolated mutantMMLV reverse transcriptase of claim
 42. 50. The composition of claim 49,wherein the reverse transcriptase comprises at least one of thefollowing mutations: a glutamic acid to lysine mutation at position E69,a glutamic acid to lysine mutation at position E302, a glutamic acid toarginine mutation at position E302, a tryptophan to phenylalaninemutation at position W313, a leucine to glycine mutation at positionL435, a leucine to methionine mutation at position L435, an asparagineto lysine mutation at position N454, an asparagine to arginine mutationat position N454, and a methionine to leucine mutation at position M651.51. The composition of claim 49, wherein the reverse transcriptasecomprises a combination of mutations selected from the following groupsof combinations of mutations: E302R/E69K/W313F/L435G/N454K;E302R/W313F/L435G/N454K; E302R/W313F/L435G; E302R/E69K/N454K;E302R/W313F; and E69K/E302R/W313F/L435G/N454K/D524N.
 52. The compositionof claim 49, wherein the reverse transcriptase further comprises aC-terminal extension.
 53. The composition of claim 52, wherein saidC-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1).
 54. Thecomposition of claim of claim 49, wherein the reverse transcriptase hasat least one of the following characteristics: increased stability,increased accuracy, increased processivity, and increased specificity.55. The composition of claim 49, wherein said reverse transcriptaselacks RNase H activity.
 56. The composition of claim 49, furthercomprising an epsilon subunit from a eubacteria.
 57. The composition ofclaim 56, wherein said epsilon subunit is from Escherichia coli.
 58. Thecomposition of claim 56, wherein said epsilon subunit is epsilon 186from Escherichia coli.
 59. The composition of claim 49, furthercomprising formamide, betaine, or DMSO.
 60. A kit comprising theisolated mutant MMLV reverse transcriptase of claim 42 and packagingmaterials thereof.
 61. The kit of claim 60, wherein the transcriptasecomprises at least one of the following mutations: a glutamic acid tolysine mutation at position E69, a glutamic acid to lysine mutation atposition E302, a glutamic acid to arginine mutation at position E302, atryptophan to phenylalanine mutation at position W313, a leucine toglycine mutation at position L435, a leucine to methionine mutation atposition L435, an asparagine to lysine mutation at position N454, anasparagine to arginine mutation at position N454, and a methionine toleucine mutation at position M651.
 62. The kit of claim 60, wherein thereverse transcriptase comprises a combination of mutations selected fromthe following groups of combinations of mutations:E302R/E69K/W313F/L435G/N454K; E302R/W313F/L435G/N454K;E302R/W313F/L435G; E302R/E69K/N454K; E302R/W313F; andE69K/E302R/W313F/L435G/N454K/D524N.
 63. The kit of claim 60, whereinsaid reverse transcriptase lacks RNase H activity.
 64. The kit of claim60, wherein said mutant MMLV reverse transcriptase further comprises aC-terminal extension.
 65. The kit of claim 64, wherein said C-terminalextension is RDRNKNNDRRKAKENE (SEQ ID NO:1).
 66. The kit of claim 60,wherein said reverse transcriptase has at least one of the followingcharacteristics: increased stability, increased accuracy, increasedprocessivity, and increased specificity.
 67. The kit of claim 60,further comprising an epsilon subunit from a eubacteria.
 68. The kit ofclaim 67, wherein said epsilon subunit is from Escherichia coli.
 69. Thekit of claim 67, wherein said epsilon subunit is epsilon 186 fromEscherichia coli.
 70. The kit of claim 60, further comprising formamide,betaine, or DMSO.